Polyol and polyether iron oxide complexes as pharmacological and/or MRI contrast agents

ABSTRACT

Iron oxide complexes, pharmacological compositions and unit dosage thereof, and methods for their administration, of the type employing an iron oxide complex with a polyol, are disclosed. The pharmacological compositions employ a polysaccharide iron oxide complex, wherein the polysaccharide is a modified polyol such as a carboxyalkylated reduced dextran. The complex is stable to terminal sterilization by autoclaving. The compositions are suitable for parenteral administration to a subject for the treatment of iron deficiencies or as MRI contrast agent. The complex is substantially immunosilent, provide minimal anaphylaxis and undergo minimal dissolution in vivo. The pharmacological compositions of the complex contain minimal free iron which can be quantified by a variety of methods.

RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.12/963,308 filed Dec. 8, 2010, which is a divisional application of U.S.patent application Ser. No. 10/410,527, filed Apr. 9, 2003, now U.S.Pat. No. 7,871,597, which is a continuation-in-part application of U.S.patent application Ser. No. 09/521,264, filed Mar. 8, 2000, now U.S.Pat. No. 6,599,498, which in turn claims the benefit of U.S. ProvisionalApplication No. 60/128,579, filed in the United States Patent andTrademark Office on Apr. 9, 1999, all of which are hereby incorporatedby reference herein.

TECHNICAL FIELD AND BACKGROUND ART

The field relates to complexes of polyols and polyethers with ironoxides including a reduced polysaccharide or derivatized reducedpolysaccharide, and methods for administering as pharmacological and/orMRI contrast agents.

BACKGROUND

Since the invention of magnetic resonance imaging (MRI), a paralleltechnology of injectable chemicals called contrast agents has developed.Contrast agents play an important role in the practice of medicine inthat they help produce more useful MRI images for diagnostic purposes.In particular, two classes of imaging agents have been developed andadopted in clinical practice. These are: low molecular weight gadoliniumcomplexes such as Magnavist; and colloidal iron oxides such as FeridexI.V.® and Combidex®. Neither of these two types of agents is ideal.Problems encountered with these agents are shown in Table 1, andinclude: expense of components; inefficiency of synthesis; loss ofcoating during terminal sterilization (autoclaving); narrow range oforgan uptake for purposes of imaging; toxic side-effects; restriction ofuse to either first pass or equilibrium dosing, and others that aredescribed herein. Agents that overcome these problems, and that combinethe properties of these two types of contrast agents, are highlydesirable.

TABLE 1 Comparison of ideal properties of MRI contrast agents withproperties of low molecular weight gadolinium based contrast agents andcolloidal iron oxides. Properties of an ideal low molecular weightcolloidal iron contrast agent gadolinium oxides Low production costs:Yes No efficient synthesis Autoclavable without Yes No excipients T1agent Yes Sometimes T2 agent No Yes Non toxic Yes No Imaging vascular NoNo compartment at early phase (as a bolus administration) and at a latestage (equilibrium phase) Multiple administration in No No singleexamination Image of multiple target Yes Sometimes organs Bolusinjection Yes No Low volume of injection No No Iron source for anemia NoYes

SUMMARY OF THE INVENTION

An embodiment in accordance with the presently claimed inventionincludes an improved method for administration of a pharmacologicalcomposition of the type employing an iron oxide complex with a polyol orpolyether, wherein the improvement comprises administering parenterallyan effective dose of an iron oxide complex with a polyol or polyether,the complex formulated in a biocompatible liquid so that uponadministration the complex provides minimal detectable free iron in asubject and minimal incidence of anaphylaxis, and effecting suchadministration at a rate substantially greater than 1 mL/min oralternatively, the administration may be at a rate of about 1 mL/sec.

We have found it possible to formulate complexes having the propertiesdescribed above. Whereas prior art complexes of dextran and iron oxidecan be made that have minimal detectable free iron, and other complexesof iron oxide may have minimal incidence of anaphylaxis, no prior artcomplexes of iron oxide have both properties. We have surprisingly founda way of providing a complex of modified polyols or polyethers with ironoxide that have both properties. We have found, for example, that apolysaccharide such as dextran, when reduced and carboxyalkylated, canbe complexed with iron oxide to produce a composition that continues(like dextran iron oxide) to have minimal detectable free iron in asubject, while (unlike dextran iron oxide) also having minimal incidenceof anaphylaxis.

Another embodiment of the present invention includes an improved methodfor administration of a pharmacological composition of the typeemploying an autoclavable reduced carboxyalkylated polysaccharide ironoxide complex with a polyol or polyether, wherein the improvementcomprises administering parenterally an effective dose of an iron oxidecomplex, the complex formulated in a biocompatible liquid so that uponadministration the complex provides minimal detectable free iron in asubject, and minimal incidence of anaphylaxis, and effecting suchadministration at a rate substantially greater than 1 mL/min oralternatively, a rate of about 1 mL/sec.

A particular embodiment of the presently claimed invention includes animproved method for administration of a pharmacological composition ofthe type employing an iron oxide complex with a polyol or polyether,wherein the improvement comprises parenteral administration of thecomplex to provide minimal detectable free iron in a subject as measuredby a catalytic bleomycin assay and minimal incidence of anaphylaxis.

Another particular embodiment includes an improved method foradministration of a pharmacological composition of the type employing aniron oxide complex with a polyol, for example dextran, or polyether, forexample polyethylene glycol, wherein the improvement further comprisesparenteral administration of the complex to provide minimal dissolutionof the complex in a human subject measured as a function of transferrinsaturation in vivo.

Still another particular embodiment provides an improved method foradministration of a pharmacological composition of the type employing aniron oxide complex with a polyol or polyether, wherein the improvementfurther comprises parenteral administration of the complex to providethe polyol or polyether complex as an immunosilent complex in a humansubject.

Another particular embodiment in accordance with the present inventionincludes an improved pharmacological composition of the type employingan iron oxide complex with a polyol or polyether, wherein theimprovement comprises formulating a polyol or polyether complexationwith iron oxide to provide upon administration to a subject minimaldetectable free iron in the subject as measured by a catalytic bleomycinassay and minimal incidence of anaphylaxis.

Yet another embodiment in accordance with the present invention is animproved pharmacological composition of the type employing an iron oxidecomplex with a polyol or polyether, wherein the improvement comprisesformulating a polyol or polyether complex with the iron oxide to provideupon administration to a subject minimal dissolution of the complex inthe subject, measured as a function of transferrin saturation in vivo.

Other embodiments include an improved pharmacological composition of thetype employing an iron oxide complex with a polyol or polyether, whereinthe improvement comprises formulating a polyol or polyether complexationwith iron oxide to provide upon administration to a subject the ironoxide complex as an immunosilent complex in a human subject.

Another embodiment in accordance with the present invention includes animproved method for administration of a pharmacological composition ofthe type employing an iron oxide complex with a polyol or polyether,wherein the improvement comprises parenteral administration of aneffective dose of the complex formulated in a biocompatible liquiddelivered at a rate substantially greater than 1 mL/min and wherein uponadministration the complex provides minimal detectable free iron in asubject, and minimal incidence of anaphylaxis; or alternatively, thecomplex is delivered at a rate of about 1 mL/sec. More particularly, theimproved method may utilize an assay for determining minimal detectablefree iron wherein the assay is any assay known in the art for measuringfree iron concentration, including a BDI assay, atomic absorptionspectroscopy, a % transferrin saturation assay, a % dialysis assay, anda bacterial growth assay. Still more particularly, the assay fordetermining minimal incidence of anaphylaxis is an ELISA assay.

In other embodiments in accordance with the invention includes animproved method for administering a pharmacological composition of thetype employing an iron oxide complex with a polyol or polyether, whereinthe improvement comprises parenteral administration of an effective doseof the complex formulated in a biocompatible liquid delivered at a ratesubstantially greater than 1 mL/min, or alternatively at about 1 mL/sec,and wherein upon administration the complex provides minimal detectablefree iron in a subject and minimal incidence of anaphylaxis, and whereinthe free iron concentration is determined using a BDI assay, and is lessthan about 750 nM, or less than about 0.04 μg/mL, or less than about0.1% of the effective dose of iron oxide, depending upon how theBDI-detected free iron measurement is reported. In alternativeembodiments, the free iron concentration is determined using atomicabsorption spectroscopy, and is less than about 1 ppm or less than about0.04 μg/mL, or less than about 0.1% of the effective dose of the ironoxide, depending upon how the atomic absorption-detected free ironmeasurement is reported; or, the free iron concentration is determinedusing a iron dialyzed % assay, and the dialyzed-determined free ironpercent is less than about 1%.

Yet another embodiment of the present invention includes an improvedmethod for administration of a pharmacological composition of the typeemploying an iron oxide complex with a polyol or polyether, wherein theimprovement comprises parenteral administration of an effective dose ofthe complex formulated in a biocompatible liquid delivered at a ratesubstantially greater than 1 mL/min, or alternatively at a rate of about1 mL/sec, and wherein upon administration the complex provides minimaldetectable free iron in a subject and minimal incidence of anaphylaxis,and wherein the improvement further comprises parenteral administrationof the complex to provide minimal dissolution of the complex in a humansubject. More particularly, in alternative embodiment, the minimaldissolution of the complex is determined using a % transferrinsaturation assay; and more particularly, the minimal dissolution of thecomplex determined by a % transferrin saturation assay is less thanabout 95% saturation for a total dose from about 1 mg/kg of body weightto about 4 mg/kg of body weight, up to a total single dose of about 500mg to about 600. An alternative embodiment further comprises parenteraladministration of the complex to provide the iron oxide complex as asubstantially immunosilent complex in a human subject. In suchembodiments, verification of administration that provides a complex thatis substantially immunosilent in a human complex may be determined by aguinea pig anaphylaxis test.

Other embodiments in accordance with the present invention include animproved pharmacological composition of the type employing an iron oxidecomplex with a polyol or polyether, wherein the improvement comprises apolyol or polyether iron oxide complex composition prepared atconcentrations of between about 1 mg/kg of body weight to about 4 mg/kgof body weight in a total volume of biocompatible liquid from about 1 mLto about 15 mL and for a total single dose from about 50 mg to about 600mg, wherein the pharmacological composition is capable of beingparenterally administered to a subject at a rate substantially greaterthan 1 mL/min, or alternatively at a rate of about 1 mL/sec, and whereinthe iron oxide complex provides upon administration minimal detectablefree iron in the subject and minimal incidence of anaphylaxis. Moreparticularly, the improved pharmacological composition may furthercomprise an iron oxide complex having minimal free iron concentration inthe subject. Determination of minimal free iron can be measured usingany standard assay for measuring free iron known in the art, including aBDI assay, atomic absorption spectroscopy, a % transferrin saturationassay, a % dialysis assay, or a bacterial growth assay. Alternatively,the improved pharmacological composition may further comprise an ironoxide complex that undergoes minimal dissolution in a human subject uponadministration to the subject. Other alternatives envision that theimproved pharmacological composition may further comprise an iron oxidecomplex that undergoes minimal dissolution upon administration in ahuman subject. Minimal dissolution may be determined using a %transferrin saturation assay. Alternatively, the improvedpharmacological composition may further comprise an iron oxide complexthat is substantially immunosilent upon administration in a humansubject, and particularly, the improved pharmacological composition mayfurther comprise an iron oxide complex that is substantiallyimmunosilent in a human subject as determined by a guinea piganaphylaxis test.

Yet another embodiment in accordance with the present invention includesa method of treating a subject with an iron oxide complex to a subjectin need thereof, the method comprising parenterally administering thecomplex formulated in a pharmaceutically acceptable formulation in abiocompatible liquid, effecting administration at a rate substantiallygreater than 1 mL/min, and providing an effective dose in the range ofabout 1 mg/kg of body weight to about 4 mg/kg of body weight in a totalvolume of biocompatible liquid of between about 1 mL and 15 mL so thatminimal free iron and minimal anaphylaxis occurs. More particularly, themethod may comprise effecting administration at a rate of between about180 L/sec and about 1 mL/min. Still more particularly, theadministration of the iron oxide complex provides minimal dissolution ofthe complex in the subject and may further provide a substantiallyimmunosilent complex to the subject. More particularly, a guinea pigtest may be used to determine that the complex administered in the abovemethod for treating is substantially immunosilent to the subject.

Another embodiment of the invention includes a method of treating asubject with an autoclavable reduced carboxyalkylated polyol, forexample dextran, iron oxide complex having at least 750 but less than1500 μmole of carboxyalkyl groups per gram of polyol to a subject, themethod comprising parenterally administering the complex formulated in apharmaceutically acceptable formulation in a biocompatible liquid,effecting administration at a rate substantially greater than 1 mL/min,and providing an effective dose in the range of about 1 mg/kg of bodyweight to about 4 mg/kg of body weight in a total volume ofbiocompatible liquid of between about 1 mL and 15 mL so that minimalfree iron and minimal anaphylaxis occurs.

Yet another embodiment includes an improved pharmacological compositionof the type employing an autoclavable carboxyalkylated polyether ironoxide complex, for example polyethylene glycol, wherein the improvementcomprises a carboxyalkylated iron oxide complex composition having atleast 250 mole but less than 1500 mole of carboxyalkyl groups per gramof polyether, prepared at concentrations of between about 1 mg/kg ofbody weight to about 4 mg/kg of body weight in a total volume ofbiocompatible liquid from about 1 mL to about 15 mL and for a totalsingle dose from about 50 mg to about 600 mg, wherein thepharmacological composition is capable of being parenterallyadministered to a subject at a rate substantially greater than 1 mL/minand wherein the iron oxide complex provides upon administration minimaldetectable free iron in the subject and minimal incidence ofanaphylaxis.

Another embodiment of the invention is a method of providing an ironoxide complex for administration to a mammal subject, the methodcomprising: producing a reduced polysaccharide iron oxide complex, andsterilizing the complex by autoclaving. In general, the reducedpolysaccharide is a reduced polymer of glucose. An example of a reducedpolymer of glucose is a reduced dextran. The reduced polysaccharide isproduced through reaction of a polysaccharide with a reagent selectedfrom the group consisting of a borohydride salt or hydrogen in thepresence of a hydrogenation catalyst. In a further aspect of the method,the iron oxide is superparamagnetic.

Another particular embodiment of the invention is a method of providingan iron oxide complex for administration to a mammalian subject, themethod comprising: producing a derivatized reduced polysaccharide ironoxide complex, and sterilizing the complex by autoclaving. According tothis method, producing the complex can include derivatizing a reducedpolysaccharide by formation of, for example, ethers, amides, esters, andamines at the hydroxyl positions of the polysaccharide. In a particularembodiment, the derivative formed is an ether of the polysaccharide,more particularly a carboxyalkyl ether of the polysaccharide, and moreparticularly, a carboxymethyl ether of the polysaccharide. Furtheraccording to this method, the reduced polysaccharide can be a reduceddextran. The derivatized, reduced polysaccharide can be isolated as thesodium salt and does not contain an infrared absorption peak in theregion of 1650-1800 cm⁻¹. In one aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 50° C. In another aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 40° C. In a further aspect of the method, the ironoxide is superparamagnetic.

In yet another embodiment, the invention provides a method offormulating an iron oxide complex coated with a reduced polysaccharide.This composition is for pharmacological use and the composition hasdecreased toxicity in comparison to a formulation of an iron oxidecomplex coated with the non-reduced polysaccharide. The method offormulating such an iron oxide complex comprises: producing a reducedpolysaccharide iron oxide complex, and sterilizing the complex byautoclaving. The formulation provides a polysaccharide which wasproduced by reacting the polysaccharide with one of a reducing agentselected from the group consisting of a borohydride salt or hydrogen inthe presence of an hydrogenation catalyst, wherein the reducedpolysaccharide iron oxide complex so made has such decreased toxicity.In a further aspect of the method, the iron oxide is superparamagnetic.

In yet another embodiment, the invention provides a method offormulating an iron oxide complex coated with a reduced derivatizedpolysaccharide. This composition is for pharmacological use and thecomposition has decreased toxicity in comparison to a formulation of aniron oxide complex coated with the non-reduced derivatizedpolysaccharide. The method of formulating such an iron oxide complexcomprises: producing a reduced derivatized polysaccharide iron oxidecomplex; and sterilizing the complex by autoclaving. According to thismethod, producing the complex can include derivatizing a reducedpolysaccharide by carboxyalkylation, for example, wherein thecarboxyalkylation is a carboxymethylation. Further according to thismethod, the reduced polysaccharide can be a reduced dextran. Thederivatized, reduced polysaccharide can be isolated as the sodium saltand does not contain an infrared absorption peak in the region of1650-1800 cm⁻¹. In one aspect of the method, producing the derivatizedreduced polysaccharide is achieved at a temperature of less thanapproximately 50° C. In another aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 40° C. In a further aspect of the method, the ironoxide is superparamagnetic.

Another embodiment of the invention provides a reduced derivatizedpolysaccharide iron oxide complex with T1 and T2 relaxation propertiesto allow contrast agent signal enhancement with T1 sequences and signaldiminishment with T2 sequences. A further aspect of the embodiment isthat the reduced derivatized polysaccharide iron oxide can beadministered multiple times for sequential imaging in a singleexamination. Yet another aspect of the agent is that it can be used toimage multiple organ systems including the vascular system, liver,spleen, bone marrow, and lymph nodes.

Another embodiment of the invention provides a reduced polysaccharideiron oxide complex for use as an intravenous iron supplement.

Another embodiment of the invention provides a reduced derivatizedpolysaccharide iron oxide complex for use as an intravenous ironsupplement.

In yet a further embodiment, the invention provides an improved methodof administering to a mammalian subject an autoclaved reducedpolysaccharide iron oxide complex. The improved method of administrationcomprising: injection of an autoclaved reduced polysaccharide iron oxidecomplex in a volume of 15 mL or less. In another aspect of theembodiment the injected volume is injected as a bolus. In a furtheraspect of the method, the iron oxide is superparamagnetic. In a furtheraspect of the embodiment the injected volume provides improved imagequality.

In yet a further embodiment, the invention provides an improved methodof administering to a mammalian subject an autoclaved derivatizedreduced polysaccharide iron oxide complex, the improved method ofadministration comprising: injection of an autoclaved reducedderivatized polysaccharide iron oxide complex in a volume of 15 mL orless. In another aspect of the embodiment the injected volume isinjected as a bolus. In a further aspect of the method, the iron oxideis superparamagnetic. In a further aspect of the embodiment the injectedvolume provides improved image quality.

An embodiment of the invention provides an improved method ofadministering to a mammalian subject a reduced polysaccharide ironcomplex to a mammalian subject wherein the improvement comprisesadministration of a reduced polysaccharide in formulation to providereduced toxicity relative to administration of a non-reducedpolysaccharide. In a further aspect of the embodiment, the iron oxide issuperparamagnetic.

An embodiment of the invention provides an improved method ofadministering to a mammalian subject a reduced derivatizedpolysaccharide iron complex in a manner that the composition providesreduced toxicity, wherein the improvement comprises utilizing a reducedderivatized polysaccharide in formulation of the composition. In afurther aspect of the embodiment, the iron oxide is superparamagnetic.

An embodiment of the invention provides a reduced polysaccharide ironoxide complex, wherein the reduced polysaccharide is derivatized, forexample, the reduced derivatized polysaccharide is a carboxyalkylpolysaccharide. The carboxyalkyl is selected from the group consistingof carboxymethyl, carboxyethyl and carboxypropyl. Further, the reducedpolysaccharide can be a reduced dextran, for example, the reduceddextran can be a reduced carboxymethyl dextran. A further aspect of thisembodiment of the invention is that the level of derivatization of thereduced dextran is at least 750 mole but less than 1500 mole of carboxylgroups per gram of polysaccharide wherein said composition has reducedtoxicity relative to composition with respect to lower levels ofderivatization.

An embodiment of the invention provides a reduced polysaccharide ironoxide complex, such complex being stable at a temperature of at leastapproximately 100° C. In a preferred embodiment, such complex is stableat a temperature of approximately 121° C. In an even more preferredaspect of the reduced polysaccharide iron oxide complex, such complex isstable at a temperature of at least 121° C. for a time sufficient tosterilize the complex. In a further aspect of the embodiment, the ironoxide is superparamagnetic.

An embodiment of the invention provides a reduced derivatizedpolysaccharide iron oxide complex, such complex being stable at atemperature of at least approximately 100° C. In a preferred embodiment,such complex is stable at a temperature of approximately 121° C. In aneven more preferred aspect of the reduced polysaccharide iron oxidecomplex, such complex is stable at a temperature of at least 121° C. fora time sufficient to sterilize the complex. In a further aspect of theembodiment, the iron oxide is superparamagnetic.

A particular embodiment of the invention is a method of formulating forpharmacological use a reduced polysaccharide iron oxide complex havingincreased pH stability in comparison to the corresponding native dextraniron oxide, the method comprising: providing dextran; and reacting thedextran with a borohydride salt or hydrogen in the presence of anhydrogenation catalyst, reacting the reduced dextran with iron salts toprovide a formulation having a stable pH.

A particular embodiment of the invention is a method of formulating forpharmacological use a reduced derivatized polysaccharide iron oxidecomplex having increased pH stability in comparison to the correspondingnative dextran iron oxide, the method comprising: providing dextran; andreacting the dextran with a borohydride salt or hydrogen in the presenceof an hydrogenation catalyst, reacting the reduced dextran with ironsalts to provide a formulation having a stable pH.

In another embodiment, the invention provides a method of formulating areduced derivatized dextran composition for pharmacological use whereinthe composition has decreased toxicity in comparison to native dextran,comprising: producing a reduced derivatized polysaccharide; andsterilizing the product by autoclaving. According to this method, thereduced polysaccharide is obtained by reacting the native polysaccharidewith one of several reducing agents selected from the group consistingof a borohydride salt or hydrogen in the presence of a hydrogenationcatalyst. In a preferred aspect of the embodiment the polysaccharide isdextran. Producing the composition can include derivatizing a reducedpolysaccharide by carboxyalkylation, for example, wherein thecarboxyalkylation is a carboxymethylation. Further according to thismethod, the reduced polysaccharide can be a reduced dextran. Thederivatized, reduced polysaccharide can be isolated as the sodium saltand does not contain an infrared absorption peak in the region of1650-1800 cm⁻¹. In one aspect of the method, producing the derivatizedreduced polysaccharide is achieved at a temperature of less thanapproximately 50° C. In another aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 40° C.

An embodiment of the invention provides an improved method ofadministering a polysaccharide to a mammalian subject, wherein theimprovement comprises parenteral administration of a reducedpolysaccharide in a formulation to provide reduced toxicity relative toadministration of a non-reduced polysaccharide.

An embodiment of the invention provides a reduced polysaccharide,wherein the reduced polysaccharide is derivatized, for example, thereduced derivatized polysaccharide is an ether-, amino-, ester-, andamido-polysaccharide. In a more particular embodiment, the reducedderivatized polysaccharide is an ether polysaccharide, more particularlya carboxyalkylether polysaccharide selected from the group consisting ofcarboxymethylether, carboxyethylether, and carboxypropyletherpolysaccharide. Further, the reduced polysaccharide can be a reduceddextran. A further aspect of this embodiment of the invention is thatthe level of derivatization of the reduced dextran is at least 750micromolar of carboxyl groups per gram of polysaccharide wherein saidcomposition has reduced toxicity relative to composition with lowerlevels of derivatization.

Another embodiment of the invention is a method of formulating a dextrancomposition for pharmacological use having decreased toxicity incomparison to native dextran, the method comprising: providing dextran;and reacting the provided dextran with a borohydride salt or hydrogen inthe presence of an hydrogenation catalyst followed by carboxyalkylation,the reduced carboxyalkylated dextran having decreased toxicity.

Another embodiment of the invention is an improved method ofadministering to a mammalian subject a derivatized polysaccharidedextran composition wherein the improvement comprises parenteraladministration of a reduced carboxyalkylated dextran in a formulation toprovide reduced toxicity relative to administration of a non-reducednon-carboxyalkylated dextran. In another aspect, an embodiment of theinvention is an improved method of administering to a mammalian subjecta derivatized polysaccharide dextran composition wherein the improvementcomprises parenteral administration of a reduced carboxymethylateddextran in a formulation to provide reduced toxicity relative toadministration of a non-reduced non-carboxymethylated dextran.

An embodiment of the invention provides a method of use of reducedderivatized dextrans as blood expanders.

The above-described embodiments are merely illustrative and not intendedto limit the invention in any way. It is envisioned that otherembodiments are possible and will be understood to fall within the scopeof the presently claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the percent of cross-reactivity observedwith compound 7228 in accordance with the present invention, as comparedto percent cross-reactivity observed with InFeD® using a ratserum-Dextran Antibody ELISA assay. The graph is plotted from the dateof Table 2.

FIG. 2 shows a Fourier transform infrared (FTIR) spectrographic analysisof carboxymethyl reduced dextran (CMRD) sodium salt obtained withExample 14.

FIG. 3 shows an FTIR spectrographic analysis of sodium salt CMRD coatedultrasmall superparamagnetic iron oxide (USPIO; see U.S. Pat. No.5,055,288) obtained in Example 43.

FIG. 4 is a graph that shows the amount of carboxymethyl groups(micromoles) per gram of product, on the ordinate, as a function of theamount of bromoacetic acid mg/gram used in reactions with reduceddextran starting material, on the abscissa. The graph is plotted fromthe data of Table 6.

FIG. 5 shows pharmacokinetics of CMRD coated USPIO in the blood of threemale rats following intravenous administration of 2.2 mg of iron per kgbody weight. Samples (0.25 mL) of blood were collected at the timesindicated on the abcissa, and relaxation times were measured on aBrucker Minispec spectrometer.

FIG. 6 shows the graph used to determine a half-life (67 minutes) ofCMRD coated USPIO in rat blood. The data of FIG. 5 were used to generatethe graph in FIG. 6. The half-life range of 61 to 75 minutes was withinthe 95% confidence level.

FIG. 7 shows MRIs of a rat, pre-administration (A) andpost-administration (B) of contrast agents, anterior portion at top.CMRD coated USPIO (5 mg of iron per kg body weight) was administeredinto the femoral vein prior to taking the post administration contrastimage. The figure illustrates enhanced visualization of the heart andsurrounding arteries and veins caused by administration of CMRD coatedUSPIO. Imaging was performed using a General Electric 2 Tesla magneticresonance imager.

FIG. 8 shows MRI images of a pig, pre-administration (A) andpost-administration (B) of contrast agent, anterior portion at top. CMRDcoated USPIO (Example 43; 4 mg of iron per kg body weight) wasadministered into the femoral vein prior to taking the postadministration contrast image. The figure illustrates enhancedvisualization of the heart and surrounding arteries and veins caused byadministration of CMRD coated USPIO. Imaging was performed using aSiemans 1.5T Magnatom Vision magnetic resonance imager.

FIG. 9 shows MRI images of the anterior portion of a normal humansubject, pre-administration (A) and post-administration (B) of contrastimaging agent. CMRD coated USPIO (4 mg of iron per kg body weight) wasadministered as a bolus into a vein in the arm prior to taking the postcontrast image. Imaging was performed 15 to 30 minutes afteradministration of contrast agent. The image illustrates enhancedvisualization of the heart and surrounding arteries and veins.

FIGS. 10A and 10B show the blood clearance kinetics in humans of imagingagent. CMRD coated USPIO (4 mg of iron per kg body weight), wasadministered as a bolus into a vein in the arm prior to taking bloodsamples. Samples were analyzed for 1/T2 relaxation to determine theblood concentration of the CMRD coated USPIO. The graph shows CMRDcoated USPIO concentration (ordinate) as a function of time (abscissa).

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

As used in this description and the accompanying claims, the followingterms shall have the meanings indicated, unless the context otherwiserequires:

“Minimal detectable free iron as measured by a catalytic bleomycinassay” means, within the context of this invention, bleomycin detectableiron (BDI) from 0 nM to about 750 nM, or from 0 μg/mL to about 0.04μg/mL, or from 0% to about 0.1% of the dose, depending upon how themeasurement is reported.

“Minimal detectable free iron as measured by atomic absorptionspectrophotometry” means, within the context of this invention, freeiron of from 0 ppm to less than about 1 ppm, or from 0 mg/g to less thanabout 0.02 mg/g, or from 0% to less than about 0.1% (mg/mL as determinedby AA, divided by total iron concentration of original sample),depending upon how the measurement is reported.

“Minimal detectable free iron as measured by an iron dialyzed % assay”is from about 0% to less than about 1%.

“Minimal dissolution of the complex, measured as a function oftransferrin saturation in vivo”, means within the context of thisinvention that at total doses from 1 mg to about 500 mg total dose,transferrin saturation remains less than about 95%.

“Minimal incidence of anaphylaxis” means, within the context of thisinvention, incidence from 0 to less than about 0.01% to about 0.05% ofacute systemic sensitivity reactions, anaphylactoid reactions or severeanaphylactic shock, including death.

“Case fatality rate” means total number of fatalities per total numberof allergic reactions, expressed as a percent.

“An administration rate of about 1 mL/sec” as used herein, is defined asa rate of 0.1 mL/sec to 3 mL/sec.

“Rapid intravenous injection” as used here in, is defined as injectionwith a delivery rate of about 1 mL/sec, as defined above.

“Biocompatible liquid” as used herein, is defined as any liquid that canbe administered to a mammalian subject and that is tolerated by themammalian subject, as evidence by a dearth of adverse reactions.Examples of biocompatible liquids include water, buffered aqueous media,saline, buffered saline, aqueous Zwitter ion solutions, low-molecularweight alcohols, amino acids, mixtures thereof, and the like.

“Heat stress” as used herein and in the accompanying claims, is definedas heating the colloid to approximately 121° C. or higher for about 30minutes at neutral pH, or other combinations of time, temperature, andpH that are well known in the art to autoclave (or terminally sterilize)an injectable drug.

“Colloid” as used in this specification and the accompanying claimsshall include any macromolecule or particle having a size less thanabout 250 nm.

Table 2 summarizes the percent cross-reactivity observed with compound7228 (C-7228) of the present invention compared to percentcross-reactivity observed with InFeD®, using a rat serum α-dextranantibody ELISA assay. This assay is used to measure the incidence ofanaphylactic response to an iron oxide complex.

Surprisingly, the iron compounds of the present invention have beenshown to exhibit superior administration profiles in a subject comparedto iron oxide compounds currently approved for either use as a hematinicagent or an MRI contrast agent. Thus, unlike existing iron oxidecomplexes currently available, polyol or polyether iron oxide complexesin accordance with the present invention, when administered parenterallyto a patient for use as a pharmacological agent, provide both minimaldetectable free iron in a subject as determined using a bleomycincatalytic assay or measured by atomic absorption spectrophotometry, andprovide minimal anaphylaxis in a patient. These iron oxide polyol orpolyether complexes may further provide minimal dissolution of the ironcomplex in a human subject as determined as a function of measurement ofpercent transferrin saturation, and may present in the human subject asan immunosilent complex to the subject's immune response system. Table 3shows standard solutions for use in calibrating an atomic absorptionspectrophotometer.

Table 4 shows free iron determination tests done on C-7228 compared toother iron oxide complexes currently available.

Table 5 shows Free Iron concentrations, in μM, μg/mL and percentage ofthe dose, for serum spiked with I.V. iron, as determined using ableomycin catalytic iron assay.

Table 1 summarizes the characteristics of two classes of MRI contrastagents that have been previously described, and shows a comparison oftheir characteristics to those of an ideal contrast agent. Agents of theinvention embody the ideal characteristics, as shown herein.

Surprisingly, the development and synthesis of preparations ofultrasmall superparamagnetic iron oxide (USPIOs) coated withpolysaccharide reduced dextrans and derivatives of reduced dextrans,such as the agents with the desirable properties as shown herein, arederived from a change in the chemical nature of one constituent, dextranT10. This change involved reduction of the terminal aldehyde group to analcohol of the polysaccharide used in its synthesis to an alcohol(Scheme 1). Scheme 1 illustrates the chemical change in a polysaccharidesuch as dextran upon treatment with sodium borohydride. The hemiacetalform of the polysaccharide (structure 1) is in equilibrium with thealdehyde form of the polysaccharide (structure 2). Structure 2represents less than 0.01% of the equilibrium mixture (Brucker, G.(1974) Organic Chemistry: Amino Acids, Peptides and Carbohydrates,Tankonykiado Press, Budapest, p. 991). Treatment of structure 2 withsodium borohydride results in its irreversible conversion to the linearpolyol form of the polysaccharide (structure 3). The dynamic equilibriumbetween structures 1 and 2 allows complete conversion, when treated withsodium borohydride, to the linear polyol (structure 3).

Dextran coated superparamagnetic iron oxide particles have particularinterest as magnetic resonance imaging (MRI) contrast agents because oftheir ability to enhance images of the liver and lymph. Feridex I.V.®(Advanced Magnetics, Inc., Cambridge Mass.) is a dextran coatedsuperparamagnetic iron oxide MRI contrast agent, and approved for use inhumans. Combidex® (Advanced Magnetics, Inc.) is a dextran coatedultrasmall superparamagnetic iron oxide (USPIO) which has completedPhase III clinical trials for both liver imaging and Phase III trialsfor lymph imaging. Combidex® has a smaller mean diameter (20 nm) thanFeridex I.V.® (60 nm), which gives it a different biodistribution inhumans. Combidex® is made by addition of base to a solution of dextran,ferric chloride and ferrous chloride. The synthetic process comprisescombining the ingredients, heating, and purifying by ultrafiltration.However, the yield of dextran added to the particles in the reaction isinefficient. Pharmaceutical grade dextran is the most expensivecomponent of the Combidex® synthesis. A more efficient use of dextran inthe synthesis of Combidex® is desirable to lower production costs.

Terminal sterilization (autoclaving) is a preferred method ofsterilizing drugs for injection. However, many superparamagnetic ironoxide colloids that are used as MRI contrast agents are synthesized withpolymer coatings and coverings that influence the biodistribution andelimination of these colloids. Upon exposure to the heat for theduration of the autoclaving process, the polymer coating can becomedissociated from the iron oxide cores. The functional consequences ofpolymer dissociation from the iron oxide are physical changes in thematerial, such as clumping, biodistribution changes (changes in plasmahalf-life), and changes in toxicity profile (potential increases inadverse events). For example, a substantial decrease in the pH of thesolution can be detected following autoclaving of iron dextranparticles, and the pH continues to fall upon further storage.

Several solutions to the problem of imparting resistance to heat stresshave been described. Palmacci et al., U.S. Pat. No. 5,262,176, herebyincorporated herein by reference, used cross-linked dextran to stabilizethe covering on the iron oxide particles prior to autoclaving. Thecross-linking process uses noxious agents such as epichlorohydrin andepibromohydrin, which must be removed from the colloid after thecross-linking reaction.

Methods of preventing clumping of the colloid induced by heat stressthat have no effect on coating dissociation have also been described.These methods generally include the use of excipients during theautoclaving process. Groman et al., U.S. Pat. No. 4,827,945, and Lewiset al., U.S. Pat. No. 5,055,288, both patents hereby incorporated hereinby reference, use citrate to prevent clumping of the particles when thecoating dissociates. However, the use of citrate in high concentrationsin combination with heat can cause toxicity. Groman et al., U.S. Pat.No. 5,102,652, hereby incorporated herein by reference, uses lowmolecular weight carbohydrates such as mannitol to prevent clumpingduring autoclaving. These excipients increase the cost and complexity ofmanufacturing the product, yet do not solve the problem of dissociationof the polymer from the iron particle.

Josephson et al., U.S. Pat. No. 5,160,726, hereby incorporated herein byreference, avoids heat stress on the coating by using filtersterilization rather than heat to sterilize the colloid. Filtersterilization is expensive since both the sterilization process andcontainer closure must be performed in a germ free environment.Additionally, filter sterilizing has a higher rate of failure than theprocess of autoclaving, which reflects the inability to obtain anenvironment for the filtration step which is entirely germ free.

Maruno et al., U.S. Pat. No. 5,204,457, describes acarboxymethyldextran-coated particle with improved stability up to 80°C. for an extended period but does not teach use of terminalsterilization by autoclaving. Hasegawa et al. (Japan J. Appl. Phys.,Part 1, 37(3A):1029-1032, 1998) describes carboxymethyl dextran coatediron particles with thermal stability at 80° C., but does not teach useof a carboxymethyl reduced dextran-coated particle, nor of terminalsterilization by autoclaving.

Magnetic resonance imaging agents act by affecting the normal relaxationtimes, principally on the protons of water. There are two types ofrelaxation, one known as spin-spin or T1 relaxation, and the secondknown as spin-lattice or T2 relaxation. T1 relaxation generally resultsin a brightening of the image caused by an increase in signal. T1processes are most useful in imaging of the vascular system. T2relaxation generally results in a darkening of the image caused by adecrease in signal. T2 processes are most useful in imaging of organssuch as the liver, spleen, or lymph nodes that contain lesions such astumors. All contrast agents have both T1 and T2 properties; however,either T1 or T2 relaxation can characterize the dominant relaxationproperty of a particular contrast agent. Low molecular weight gadoliniumbased contrast agents are T1 agents, and have primary application in theimaging of vascular related medical problems such as stroke andaneurysms and the brain. Iron oxide based colloidal contrast agents areT2 agents, and have primary application in imaging tumors of the liverand lymph nodes (prostate and breast cancer). An agent possessing bothT1 and T2 properties would be desirable. Using such an agent would (I)provide a single drug for all applications, and simplify the inventoryof the pharmacy, (ii) simplify imaging in the MRI suite, and (iii)improve patient care by permitting simultaneous examination of multiplemedical problems in a single patient during a single examination, ratherthan requiring use of either a T1 or a T2 contrast agent.

Information regarding anatomical features within the vascular system canbe obtained using contrast agents in two ways. When the contrast agentis first administered as a bolus, it initially passes through thevascular tree as a relatively coherent mass. Coordinating the time ofimaging of the desired anatomical feature to the time when the boluspasses through that feature can provide useful information. Thistechnique of contrast agent use is called first pass imaging. At a latertime, the bolus has been diluted by mixing, and attains an equilibriumconcentration in the vascular system. Under certain circumstances, thisequilibrium or steady state can offer useful information. Imaging can beperformed at an early phase, within minutes after injection of thecontrast agent (“first pass”), and at a later phase, from about tenminutes after injection of the contrast agent (equilibrium phase).Gadolinium agents are suited only for first pass imaging due to theirready diffusion from the vascular system into the interstitial spaces ofthe tissues. Previously described colloidal iron oxides are useful forthe equilibrium due to their requirement for dilute administration overa prolonged time period. Colloidal iron oxides do not leak into theinterstitial space but can remain in the vascular system for hours. Anagent offering the opportunity to perform both first pass imaging andequilibrium imaging would be desirable.

During administration in a medical setting of a contrast agent for“first pass” imaging, the timing of imaging and passage of the “firstpass” of the contrast agent may not coincide. If a useful image was notobtained, it becomes desirable to administer a second dose of contrastagent to obtain another “first pass” image. On other occasionsradiologists find it useful to examine several volumes within thepatient requiring a multiple dosing regimen of contrast agent in orderto obtain “first pass” images at each of multiple sites of interest.With gadolinium contrast agents, this multiple administration “firstpass” application is not possible because the gadolinium leaks out ofthe vascular space producing a fuzzy background around blood vessels ofinterest. Current iron oxide colloidal based contrast agents are notsuitable as they are administered not as a bolus, but as a dilutesolution over a long time, obviating “first pass” applications.

Diagnosis of tumor progression in cancer patients is important forcharacterizing the stage of the disease, and for assessing treatment. Tominimize cost and discomfort to the patient, it is desirable in an MRIexamination to administer a single dose of contrast agent that wouldallow assessment of multiple organ systems that might be affected by thedisease. For instance, in primary breast cancer, it is desirable toassess tumor status in the breast and at multiple metastatic sitesincluding the liver, spleen, bone marrow, and lymph nodes.Administration of gadolinium based contrast agents can not satisfy thisrequirement due to their short half-life in the body, their leakage intothe vascular system, and their inability to concentrate within organs ofinterest. Iron oxide colloid based contrast agents such as Combidex® canserve in this multiple capacity while Feridex I.V., another iron oxidecolloid contrast agent, is limited to imaging the liver and the spleen.

Administration of a contrast agent in a small volume (less than 5 mL) isdesirable, as small volume administration improves the resolutionobtained from first pass imaging, and minimizes injection time anddiscomfort to the patient. Gadolinium based contrast agents areadministered in volumes of about 30 mL due to constraints caused by thesolubility and potency of these agents. Currently, iron oxide basedcontrast agents are administered as a dilute solution in a large volume(50-100 mL) over an extended period of time (30 minutes). Theseconstraints arise from safety issues associated with the rapid andconcentrated administration of iron oxide based agents. Bolus injectionis desirable in that it allows first pass imaging and shortens contacttime between the patient and health care provider. Further bolusinjection allows the practitioner to administer the contrast agent whilethe subject is in the MRI apparatus during the examination, therebyoptimizing efficient use of instrument imaging time. Gadolinium basedagents can be administered as a bolus.

Gadolinium based contrast agents consist of a chelating molecule and thegadolinium cation. Gadolinium is a toxic element and must be excretedfrom the body to avoid toxicity. Colloidal iron oxides are not excretedfrom the body but are processed in the liver and other organs tometabolic iron, such as the iron in hemoglobin. Thus, compositions ofthe invention can serve as an iron supplement for patients sufferingfrom anemia, and are especially useful for patients undergoing treatmentwith erythropoietin. However, of the four general types of iron oxidecompounds used as MRI contrast agents and/or hematinic agents (ironsucrose, iron gluconate, iron dextran, and the iron oxides of thepresent invention) all but those of the presently claimed invention haveserious drawbacks making them less than desirable for their intendedpharmacological use.

For example, two of the oldest iron compounds administered as hematinicagents to patients suffering from acute anemia are iron gluconate andiron sucrose, examples of which include Ferrlecit® and Venofer®,respectively. These iron compounds were developed to overcome theproblem of iron toxicity observed upon injection of iron salts into asubject (see C. W. Heath et al., “Quantitative Aspects of IronDeficiency in Hypochromic Anemia,” J. Clin. Invest. 11(6) (1932), pp.1293-1312). Compounds such as Ferrlecit® and Venofer® were somewhatsuccessful in reducing free iron concentrations, the substance primarilyresponsible for the iron toxicity observed with iron salts. They werelimited in dosage amounts and rates of administration, however, becauseof problems associated with serious side effects such as hypotensionwhich arose in a significant number of patients. For example, at dosagesof Ferrlecit® higher than the maximum recommended dose of ˜125 mg pertreatment (either diluted to 100 mL and given over the course of 1 hour,or delivered undiluted as a slow intravenous injection over the courseof 10 minutes) ten to thirty percent of the patients treated sufferedsevere nausea and vomiting, hypotension, and other side effects. See B.Bastani et al., “Incidence of Side-Effects Associated with High-DoseFerric Gluconate in Patients with Severe Chronic Renal Failure,”Nephrol., 8 (2003) pp. 8-10.

Other iron compounds were then developed to overcome the limits indosage amounts and administration rates associated with iron gluconateand iron sucrose compounds, while still maintaining low free ironconcentrations to avoid iron toxicity. One such compound is irondextran, an example of which is INFeD®. Unfortunately, although irondextran did allow administration of larger dosages, if given slowly, arelatively high incidence of anaphylaxis was observed, including fatalreactions. For example, from 1976-1996 in the United States, 196allergic/anaphylactic reactions were reported with administration ofiron dextran, and of those 196 cases, 31 were fatal—an unacceptablefatality rate of 15.8% of all allergic reactions observed (see Bastaniet al. at 9).

The risk of a fatal response is seen as so great that Watson Pharma,Inc., the manufacturer of INFeD®, is required by the FDA to include a“black box” warning, the highest level of warning required by the FDA,on the drug information sheet that accompanies the packaging of the drug(see Exhibit A, attached hereto). Other references show the incidence ofside effects for INFeD®, Dexferrum®, Ferrlecit® and Venofer® (e.g. seeExhibit B, attached hereto—“The Drug Monitor, Parenteral Iron (IV)Supplementation, by Nasr Anaizi, Ph.D.). All exhibit side effects in arelatively high percentage of patients treated, from hypotension (whichis sometimes severe), nausea, and diarrhea, to chills, headache, andfever, among others.

In addition, dextran alone can also sometimes elicit a fatalanaphylactic response when administered intravenously (i.v.) in man(Briseid, G. et al., Acta Pharmcol. et Toxicol., 1980, 47:119-126;Hedin, H. et al., Int. Arch. Allergy and Immunol., 1997:113:358-359).

For compounds such as Ferrlecit® and Venofer®, and to a lesser extentINFeD®, the presence of free iron upon in vivo administration to a humansubject is undesirable for reasons other than the reported side-effectsof hypotension, nausea and diarrhea. Studies have shown that in vivofree iron is undesirable because it fosters the growth of bacteria andmay even lead to increased risk of death. See Collins et al., J. Am.Soc. Nephrol. (1997) 8, pp. 190A (Abstract); Bullen, Rev. Infect. Dis.(1981) 3, pp. 1127-1138; and Weinberg, Microbiol. Rev. (1984) 64, pp65-102. In addition, Parkinnen et al., in Nephrol. Dial. Tansplant,2002, 15, pp. 1827-1834 measured free iron using a bacterial growthassay where the bacteria is S. epidermis. This assay is based on theknowledge that S. epidermis (and by implication, other Gram-negativebacteria) cannot utilize transferrin-bound iron. Parkinnen et al. showedthat serum samples with calculated transferrin saturation of >80% werepositive for both a BDI assay and the bacterial growth assay, whereasnone of the samples with calculated transferrin saturation levels of<80% were positive for the BDI assay, although a few such samples didshow slow sustained growth after a considerable lag period (seeParkinnen at 1833, col. 1, third paragraph).

Iron-induced impairment of neutrophil function, leading to decreasedphagocytosis has also been reported in vitro (vanAsbeck et al., J.Immunol. (1984) 132, pp. 851-856) and in vivo in patients supplementedwith i.v. iron (Patruta et al., J. Am. Soc. Nephrol. (1998) 9, pp655-663). And lastly, infusion of ferric saccharate administered atdoses regularly given for intravenous iron supplementation results in agreater than 4-fold increase in non-transferrin bound iron and alsocauses transient reduction of flow-mediated dilatation, indicative ofendothelial dysfunction (see Rooyakkers et al, Eur. J. Clin. Inv. 2002,32, pp 9-16).

Overall, these findings indicate that i.v. iron dosage regimens whichmay lead to transferrin oversaturation should be avoided. As such, it isan embodiment of the invention that a polyol or polyether iron oxidecomplex in accordance with the present invention, when administeredparenterally to a patient for use as a pharmacological agent, providesboth minimal detectable free iron in a subject as determined by a BDIassay or by atomic absorption spectrophotometry or by a bacterial growthassay, and minimal anaphylaxis in a patient. In another embodiment, theiron oxide polyol or polyether complexes further provide minimaldissolution of the iron complex in a human subject, determined as afunction of percent transferrin saturation, and in another embodiment,the polyol or polyether iron oxide complex presents in the human subjectas an immunosilent complex.

An embodiment of the invention provides a method for the synthesis of acolloid of an iron oxide associated with a water soluble polyol orpolyether coating in a manner that mitigates dissociation of the coatingfrom the iron oxide when the material is subjected to heat stress, orwhen administered parenterally to a patient as a pharmacologicalformulation.

A method that is an embodiment of the invention includes the steps oftreating a polysaccharide with a reducing agent such a borohydride saltor with hydrogen in the presence of an appropriate hydrogenationcatalyst such Pt or Pd to obtain the reduced polysaccharide, such thatthe terminal reducing sugar has been reduced to give an open chainpolyhydric structure. The reduced polysaccharide may be anarabinogalactan, a starch, a cellulose, an hydroxyethyl starch (HES), aninulin or a dextran.

Moreover, it is an embodiment of the invention that a polyol orpolyether, including the reduced polysaccharide described above or apolyethylene glycol, may be further functionalized prior to particleformation. Such a method further comprises mixing the polyol, reducedpolysaccharide, or polyether with iron salts in an acidic solutionselected from the group comprising ferric salts, ferrous salts, or amixture of ferrous and ferric salts, cooling the solution, neutralizingthe solution with a base, and recovering the coated iron oxide colloid.

In accordance with a further embodiment of the invention, the baseswhich may be employed are sodium hydroxide, sodium carbonate and morepreferably, ammonium hydroxide, for the step of neutralizing thecolloid. In one particular embodiment of the invention, thepolysaccharide derivative is reduced dextran and the iron salts may beferrous and ferric salts, which produce a superparamagnetic iron oxidecolloid with a water soluble coating that does not dissociate from theiron oxide core under heat stress during terminal sterilization.

In another embodiment of the invention, only ferric salts are employed,yielding a non-superparamagnetic particle.

In another embodiment, a coated colloid may be prepared by adding apolyol or polyether to an iron oxide sol (a colloidal dispersion in aliquid), adjusting the pH to 6-8 and recovering the coated iron oxidecolloid.

The iron oxide polyol or polyether colloids in accordance with theinvention have substantially improved physical characteristics andmanufacturability compared to previously described materials. Improvedphysical characteristics are evident in the ability of the colloid towithstand heat stress, as measured by subjecting the colloid to atemperature of 121° C. for 30 minutes. Colloid particles made accordingto the invention show less evidence of polyol or polyether dissociationunder stress, remaining colloidal, and exhibiting no appreciable changein size.

An example of a colloid with an unstable polysaccharide coating includesCombidex®, which when subjected to heat stress, lost 43% of its dextrancoating, and increased in particle diameter size from 21 nm to 587 nm;significant clumping of material was observed upon visual analysis.Another superparamagnetic iron oxide dextran colloid, Feridex, preparedaccording to U.S. Pat. No. 4,770,183, also exhibited increased particlesize, as demonstrated by the inability of the heat treated colloid topass through a filter having a 0.8 μm pore size, after a heat treatmentcomprising only 30 minutes at 121° C.

An example of a colloid that provides more than minimal dissolution ofthe complex upon parenteral administration to a subject is irongluconate and iron sucrose.

During manufacture, a process that is an embodiment of the inventiontypically uses one tenth or less the amount of polysaccharide comparedto the amount required in previous preparations using non-reducedpolysaccharide, resulting in substantial raw materials cost savings dueto the improved efficiency of the process of the invention.

Variation in such factors as the nature of the polyol or polyetherderivative (i.e ethers, esters, amides, amines, carboxyalkylethers,)concentration, polyol or polyether concentration, or base concentrationand/or Fe(III)/Fe(II) concentration can produce colloids with differentmagnetic susceptibilities and sizes. Changing the Fe(III)/Fe(II) ratioschanges the particle size and alters the magnetic susceptibility. Higherratios (for example, 2.0 mol/mol) tend to decrease susceptibility,whereas lower ratios (for example, less than 1.5 mol/mol) tend toincrease particle size.

The process may be adjusted to yield colloids with different biologicalproperties by changing the type of polyol or polyether and furtherderivatizing the particle after synthesis, such as by etherification,esterification, carboxyalkylation, amidation, or amination, for example.

The colloids that are an embodiment of the invention can be used ascontrast agents for magnetic resonance imaging (MRI) or in otherapplications such as magnetic fractionation of cells, immunoassays,magnetically targeted drug delivery, and as therapeutic injectable ironsupplements. These colloids are particularly suited to parenteraladministration, because the final sterilization typically isautoclaving, a preferred method since it eliminates viability of allcellular life forms including bacterial spores, and viruses.

Previous methods for making colloids required the addition of excipientssuch as citrate or low molecular weight polysaccharides as stabilizersduring the autoclaving process (see U.S. Pat. No. 4,827,945 and U.S.Pat. No. 5,102,652), or avoided heat stress altogether by use of filtersterilization (see U.S. Pat. No. 5,150,726). Thus, the embodiments ofthe present invention that are the methods for synthesizing thecolloids, and the embodiments of the present invention comprising thecolloid compositions, provide utilities as significantly improved MRIcontrast agents, and hematinic agents that are iron supplements. Theimprovements provided in these agents over prior art are found in thefollowing facts demonstrated in the examples herein: that the agentswhich are embodiments of the present invention are heat sterilizable byautoclaving, and are thus optimized for long-term storage at ambienttemperatures; that these agents do not require the addition ofexcipients for maintenance of stability during the sterilization orstorage processes; that the agents are non-toxic to mammals includinghumans; that an effective dose of the agents used for imaging is asmaller amount of material than the agents described in the art; andthat the pharmacokinetics following administration are such thatiterated successive doses administered after a brief interval afteradministration of a first dose can be used to obtain additional imagesduring a single clinical visit and use of the imaging apparatus.

In the case of polyols such as dextran and derivatives thereof, orpolyethers such as polyethylene glycol and derivatives thereof, theformulations prepared by this method are less immuno-responsive inmammals, as shown by data obtained using a rat model, and in clinicaltrials in human subjects. The dextran- and dextran derivative-coatediron particles enhanced imaging of the heart, lungs, kidneys, and otherorgans and systems in three mammalian species: rat, pig, and human. Thedextran- and dextran derivative-coated iron particles can be used alsoas hematinic agents, to provide iron in a more efficiently absorbedformat than is true of oral iron supplements, to groups of patients whoare chronically iron-deprived, such as dialysis patients, cancerpatients, gastroenteritis patients, and recipients of erythropoietin.ELISA assays can also verify that administration of iron oxide polyol orpolyether complexes in accordance with the present invention result inminimal cross-reactivity with a rat serum a-dextran antibody, as isshown in Table 2 and FIG. 1 and described in Example 1 for a polyol ironoxide complex, indicating that such compounds present as immunosilentcomplexes in a subject, and thus provide minimal incidence ofanaphylactic response.

The derivatized reduced dextrans can be used also as plasma extenders,which, unlike blood and blood fractions, do not have to be cross-matchedimmunologically, and unlike human serum albumin preparation, can besterilized in a manner that destroys viruses, including strains ofhepatitis, CMV, and HIV, spongiform encephalitis, and other infectiousagents. The plasma extenders of the invention do not have to berefrigerated or stored away from light and heat, and are thusadvantageous in emergency medical situations, such as treatment of shockdue to loss of blood such as trauma, even in tropical climates.

Example 1 describes the method for performing the ELISA assay as reliedon in FIG. 1 and Table 2.

Examples 2 and 3 describe methods for measuring free iron in a sampleafter administration of a polyol or polyether iron oxide complex inaccordance with the present invention. The results of these measurementswith a polyol iron oxide complex are shown in Tables 3, 4, and 5.

Example 4 describes a method for measuring dissolution of the complex ina subject, as a function of transferrin saturation.

Example 5 describes a method for preparing a polyol or polyether ironoxide complex in accordance with the present invention.

Examples 6 and 7 describe improved methods for parenteral administrationof a polyol or polyether iron oxide complex in a subject to provideminimal detectable free iron, as measured by a bleomycin catalytic assayand minimal anaphylactic response, as indicated by cross-reactivity witha rat serum α-dextran antibody and as determined by volume of paw edema.

Examples 8 and 9, respectively, describe improved methods forformulating an iron oxide complex with a polyol or polyether whereinupon administration at a delivery rate from to a subject the complexexhibits minimal dissolution in the subject, measured as a function oftransferrin saturation in vivo, or whereupon administration to asubject, the complex is immunosilent in the human subject.

Examples 10, 11 and 12 show the methods for making reduced dextrans oftype T1, T5, and T10, respectively. Example 13 describes preparation ofreduced pullulan.

Examples 14-18 describe the synthesis of carboxymethyl reduced dextranT10 with varying degrees of carboxymethylation, from native dextran T10(Table 6).

Examples 19-24 describe the synthesis of carboxymethyl reduced dextranT10 with varying degrees of carboxymethylation, starting with reduceddextran T10 (Table 7).

Examples 25-25 describe the synthesis of carboxymethyl dextran T10, T40,and T70 from native dextran.

Examples 28-35 describe the preparation of reduced and native dextrancoated iron oxides. The conditions of the reactions in these exampleswere chosen to yield USPIOs coated either with reduced or non-reducedpolysaccharides. The reactions conditions for the native dextran ironoxide preparations were the same as for the reduced dextran preparationsof the same molecular weights, to allow comparison of the effectivenessof the respective dextrans in coating particles. Mean volume diameter(MVD) and magnetic susceptibility of iron oxide preparations obtainedusing reduced in comparison to native polysaccharides (prepared in theseexamples) are summarized in Table 8.

Examples 35, 38, and 40 and 37, 29, and 41 describe procedures for thepreparation of USPIOs with native T1, T5, and T10 dextrans or withPEG4000, PEG8000 or PEG10000, respectively, to obtain iron oxidecolloids having a particle diameter of less than 30 nm. A comparison ofeffects of native dextrans (Examples 36-40) and their respective reduceddextrans (Examples 28, 30, and 32) in the synthesis and properties ofiron oxide colloids is shown in Table 9.

Examples 42-43 describe the preparation USPIOs coated with carboxymethylnative dextran T10 and carboxymethyl reduced dextran T10.

Examples 44-53 describe the preparation of USPIOs coated withcarboxymethyl reduced dextran T10 preparations containing varyingextents of carboxymethylation. The effect of extent ofcarboxymethylation of CMRDs on colloid size of USPIOs is shown in Table10. The effect of extent of carboxymethylation of CMRDs on solubility offerric/ferrous chloride solutions is shown in Table 11.

Examples 54-60 describe the synthesis of iron oxide sols and theirstabilization with native and reduced dextrans and CMRD. Example 61describes preparation of CMRD coated non-magnetic iron oxide colloidusing base precipitation of ferric chloride and CMRD.

Example 62 examines the effect of the process of sterilization byautoclaving of various preparations of USPIOs coated with reduced andnative dextrans on the properties of these particles. The results areshown in Tables 12 and 13.

Example 63 reports the relaxation properties of various contrast agentscomparing these properties for gadolinium based contrast agents andUSPIOs prepared with native dextran and carboxymethyl reduced dextranT10 (Table 14).

In Examples 64-67, the presence of symptoms of toxicity to rats ofreduced and non-reduced (native) dextran coated USPIOs was determined,with response to an anaphylactic type reaction. The extent of theanaphylactic type reaction is determined by volume of paw edema. Similarstudies were performed using native, reduced, and carboxymethylatedreduced dextrans. The results are summarized Tables 15-18.

Examples 65 and 66 describe a rat paw volume edema test and guinea piganaphylaxis test, used to determine the likelihood of anaphylacticresponse to parenteral administration of polyol or polyether iron oxidecomplexes in a human subject.

Example 67 describes toxicity studies in rates of reduced andnon-reduced dextran.

Example 68 and FIGS. 5 and 6 show the kinetics of clearance of a CMRDcoated USPIO from rat circulation. The half-life of the agent isdetermined.

An enhanced MRI scan is shown in Example 69 and FIG. 7 followingadministration of CMRD coated USPIO, the scan showing images of the ratheart, aorta and other cardiac-associated arteries. Example 70 and FIG.8 show a CMRD coated USPIO enhanced MRI scan of the anterior portion ofa pig. Example 71 shows that injection of CMRD coated USPIOs into humansubjects, as part of a clinical trial, produced no adverse effects.Example 72 describes the biodistribution (FIG. 9), imaging kinetics(FIG. 10 and Table 19), and absence of background in MRI usage of thismaterial in humans. The data in this example show the ability of thepractitioner of the invention to perform multiple administrations andobtain subsequent images within the real time of an office visit orvisit to a MRI facility.

EXAMPLES Example 1 ELISA Assay

Plastic microplates were pre-coated with a dextran-BSA conjugate. Toeach microwell was added 100 microliters of IV-Fe at serial dilutionconcentrations, followed by 100 microliters rat serum containingantibodies against dextran. The plate was incubated for 1 hour at roomtemperature, decanted and washed with 0.2% Tween 80 solution in water.To each well was added 200 microliters of anti-rat IgG-alkalinephosphatase conjugate. The plate was incubated for 1 hour at roomtemperature, decanted and washed with 0.2% Tween 80 solution in water.To each well was added 200 microliters of p-nitrophenylphosphate. Theplate was incubated at room temperature for 8 minutes, and theabsorbance of the wells was read at 405 nm. Samples of dextran T70 andthe IV-Fe were run at the same time. ED50 values were calculated usingthe Molecular Devices Microplate reader software.

Significantly, C-7228, when administered parenterally to a subject,provides minimal anaphylaxis. Using an ELISA assay to measurecross-reactivity of intravenous iron versus dextran as the method fordetermining anaphylactic response, it was observed that at 730 mg Fe/mLfor compound 7228 only 11% cross-reactivity was detected, compared to467% cross-reactivity for InFeD® at only 15 mg Fe/mL (Table 2 andaccompanying FIG. 1).

As can be seen in Table 2 and FIG. 1, C-7228, even at levels of 730 μgof Fe/mL dosage resulted in only 11% cross-reactivity, whereas INFeD® atonly 15 μg Fe/mL resulted in 467% cross-reactivity. At comparable levelsof C-7228 to those of INFeD® (i.e. C-7228 at 15 μg Fe/mL)cross-reactivity with C-7228 was undetectable.

TABLE 2 ELISA data showing levels of cross-reactivity with rat serumα-dextran antibodies for C-7228 compared to INFeD ®. ELISA — — DextranT10 — — — Coated Plate Rat Serum — — — α-Dextran Antibody — — — — ED50T70 ED50 cross Inhibitor μg Fe/mL μg/mL reactivity — — — 7228 730 77 11%INFeD ®  15 70 467% 

Example 2 Free Ionic Iron Determined by Atomic Absorption

Atomic absorption (AA) spectrophotometry is used to determine free ioniciron in samples filtered through a 30-K molecular weight cut-offmicropartition membrane.

Materials

AA Spectrophotometer (Perkin Elmer 3100 or equivalent) wavelength 296.7nm, equipped with iron lamp and air/acetylene flame.

5 ppm, 10 ppm, 15 ppm and 20 ppm Iron standards (NIST traceable)

Pipets/tips

Purified water

Centricon 30-K membrane filters—AMI Code 1582

Tabletop centrifuge with fixed angle rotor capable of 1000-5000×g

Standard Preparation

Working standards are prepared according to the following dilutionsshown in Table 3.

TABLE 3 Standard Amount of 1000 ppm Concentration Standard per Liter  5ppm  5 mL 10 ppm 10 mL 15 ppm 15 mL 20 ppm 20 mLSample Preparation

Each sample is mixed by preparing an accurate 1:10 volumetric dilutionof each sample in purified water in a microconcentrator tube bypipetting 200 μL of the sample into the tube and adding 1.8 mL of water.For each dilution, the sample is dispersed with a pipet tip and thedilution factor (D_(i)=10) is recorded.

Filled Amicon tubes are centrifuged at 1000×g at room temperature for 30minutes with care taken to not exceed the recommended centrifuge speedfor Centricon 30-K membrane filters. Each eluent is then diluted 1:2with purified water in a test tube to obtain sufficient volume for AAdetermination. The second dilution is then recorded on the data sheet asD_(e) and each sample dilution is then mixed before reading an AA.

Assay

The atomic absorption spectrophotometer is set up according to theparticular instrument instructions. In the case of a Perkin-Elmer 3100spectrophotometer, the instrument has a single element iron hollowcathode lamp, an oxidizing lean, blue air-acetylene flame, a flowspoiler, and a stainless steel nebulizer. The instrument settings arethen: spectral line, 296.7 nm; slit width, 0.2 nm; integration time, 1.0seconds; and readings per sample, 10. The instrument is used with abackground correction enabled, if available, with the results calculatedin ppm by the instrument.

The instrument is first calibrated using zero (purified water) and 20ppm standards. Continue calibrating the instrument with controls of 5,10 and 15 ppm which are measured and read. Results must be withinspecified ranges. Once the AA spectrophotometer is properly calibrated,the iron concentration for each test sample solution is measured. Thesampling tube is rinsed with purified water between each sample, whichacts to reset the automatic zero on the instrument.

Calculations

For AA readings of 1.0 ppm or less, the final value is reported as “lessthan” the calculated concentration of 1 ppm reading (P=1 in the formulabelow). The ionic iron content in mg/mL is then calculated using thefollowing formula:

${{mg}\text{/}{mL}} = {\frac{P \times D_{i} \times D_{e}}{1000\mspace{14mu} µ\; g\text{/}{mg}}\mspace{14mu}{where}}$P = Value  from  AA  (ppm)(µ g/mL)D_(i) = Initial  sample  dilution  factor  ( = 10)D_(e) = Eluent  dilution  factor  ( = 2)

Using this formula, the final value is then less than 0.02 mg/g for AAreadings of 1.0 ppm or less.

The percent ionic iron, as mg/mL obtained in this assay divided by thetotal iron concentration of the original sample, can then be calculated.For AA readings of 1.0 ppm or less, the value is reported as <0.1%. Asshown in Table 4, results from quality control tests done on an ironcomplex in accordance with the present invention—Code 7228 (C-7228)—arecompared to the results of the same tests done on Ferrlecit® (irongluconate), Venefer® (iron sucrose), InFeD® (iron dextran), andDexferrum® (iron dextran). As can be readily seen, compound 7228produces only 0.001% free iron in solution (when measured using atomicabsorption spectrophotometry on samples filtered through a 30-K filtermolecular weight cut-off micropartition membrane filter) as compared to2.36% for Ferrlecit®, 0.038% for Venofer®, 0.29% for InFeD®, and 0.000%for Dexferrum®. In another measurement to show free iron, listed as irondialyzed % in the R&D tests, C-7228 shows 0% compared to 3.7% and 1.8%for Ferrlecit® and Venofer®, respectively. C-7228 also contains zeroparticulates ≧10 μL and just one particulate ≧25 μL, compared to 6≧10 μLand 7≧25μ for Ferrlecit®, for example.

TABLE 4 Tests Comparing C-7228 to Other Iron Oxide Complexes CurrentlyAvailable C-7228 Ferrlecit ® Venofer ® INFeD ® Dexferrum ® Free iron(30K 0.001% 2.360% 0.038% 0.298% 0.000% Filter Iron dialyzed %    0% 3.7%  1.8%    0%    0% (corrected for control)

Example 3 Free Iron Determined by a Bleomycin Detectable Iron (BDI)Assay

Another sensitive assay for determining free iron concentration is acatalytic bleomycin assay referred to as the BDI assay. This standardassay uses the anti-tumor compound bleomycin, which requires catalyticamounts of iron for activity, to detect the presence of iron in asolution, with the ability to detect free iron concentrations below 50nm (see FIG. 2 and throughout in M. J. Burkitt et al., Clin. Sci. (2001)100, pp. 238-247 hereby incorporated by reference herein).

There are two common versions of the BDI assay, one utilizing changes inethidium bromide-enhanced fluorescence of DNA to determine free ironconcentrations, (see Burkitt, supra) and the other utilizing measurementof DNA damage through formation of malondialdehyde (MDA), which thenforms a chromophore with thiobarbituric acid (TBA) known as the(TBA)-2-MDA adduct which can be measured by ultraviolet spectroscopy at532 nm (see J. M. Gutteridge et al. (1987) Life Chem. Rep. 4, 113-142and P. J. Evans et al., (1994) Methods Enzymol. 233, 82-92 both of whichare hereby incorporated by reference herein).

Table 5 shows the results of a BDI assay done to measure intravenous(I.V.) free iron concentrations in solutions of Ferrlecit®, Venofer®,and INFeD® as compared to C-7228. As can be seen below in Table 5, totaliron administered for the different iron complexes ranged from 578 to610 μM, or 32.3 to 34.1 μg/mL, depending on the units reported. However,bleomycin-detectable iron (BDI) was lowest for C-7228 and highest forFerrlecit®, whether reported as μM, μg/mL, or % of dose delivered.

TABLE 5 Catalytic Bleomycin Assay showing Bleomycin Detectable Iron(BDI) for solution of known iron oxide complexes Ferrlecit ®, Venofer ®,and INFeD ® compared to C-7228. I.V.-Fe Total Fe Catalytic Fe (BDI) μMμg/mL μM μg/mL % of dose Ferrlecit ® 578 32.3 8.19 0.457 1.42% Venofer ®610 34.1 4.3 0.240 0.70% INFeD ® 574 32.1 1.18 0.066 0.21% C-7228 56631.6 0.48 0.027 0.08%

Example 4 Determination of the Dissolution of C-7228 and Other Polyol orPolyether Iron Oxide Complexes as a Function of Transferrin SaturationIn Vivo

Transient measurements of transferrin “oversaturation” (i.e. greaterthan 100% saturation) have been reported after intravenous treatmentwith iron gluconate (such as Ferrlecit®) and iron sucrose (such as)Venofer®, see Kooistra et al., Eur. J. Clin. Invest. (2002) 32(Suppl. 1) pp. 36-41, which is hereby incorporated by reference herein.Such measurements are indicative of iron oxide complexes thateffectively fall apart in vivo. Compounds in accordance with the presentinvention show minimal dissolution after i.v. treatment in subjects, asindicated by transferrin saturation. In accordance with embodiments ofthe present invention, iron oxide complex dissolution is determined as afunction of transferrin saturation, or oversaturation. Procedures formeasuring % transferrin saturation are well-known in the art, and aredescribed in Singh et al., Anal. Biochem. (1990) 186, pp. 323; Kooistraet al., Eur. J. Clin. Inv. (2002) 32, pp. 36-41; and Parkinnen et al.,Nephrol. Dial. Transplant (2000) 15, pp. 1827-1834 at pp. 1829-1830, theentire contents of which are all hereby incorporated by referenceherein. In addition, using a procedure outlined in Kooistra et al. (seeabove), the concentration of total plasma iron binding sites can bedetermined using a calculation based on serum transferrin concentration.

Transferrin iron binding capacity is calculated as follows (using themolecular weight of transferrin to be 79.57 kD and 1 mol of transferrinequals 2 mol of iron binding sites):

Transferrin Ironbinding capacity (μmol/L)=serum transferrin (g/L)×25.14

Thus, when all iron binding sites in plasma derive from transferrinmolecules,

${{Transferrin}\mspace{14mu}{saturation}} = \frac{{serum}\mspace{14mu}{iron}\mspace{14mu}\left( {{µmol}\text{/}L} \right)}{{transferrin}\mspace{14mu}{iron}\mspace{14mu}{binding}\mspace{14mu}{capacity}\mspace{14mu}\left( {{µmol}\text{/}L} \right)}$

Therefore, it is possible to determine whether an iron oxide complexundergoes dissolution upon i.v. administration by determining the %transferrin saturation. In particular, a % transferrin saturation ofover 100% (oversaturation) is strongly indicative of a complex thatundergoes rapid and extensive dissolution in vivo.

Example 5 Preparation of Carboxyalkyl Polyether Compounds

The following solutions are prepared and cooled to 5° C.: Solution A:10.5 N sodium hydroxide in water; Solution B: 4.34 M haloalkylcarboxylicacid; and Solution C, 75.7 g of polyether ethylene glycol in 187.5 mLwater.

Solution A (150 mL) was added to Solution C all at once. After 5 min,145 mL of Solution B is added and the combined solution is then stirredfor 120 min while the temperature is maintained between 20° C. and 25°C. The mixture is neutralized with 6 M HCl, passed through a 0.2-μm poresize filter, and diluted to 2 liters. The product is purified byrepeated ultrafiltration against a 3-kDa MWCO ultrafiltration membrane,0.2 μm filtered again, and lyophilized. The yield is then determined andthe recovered solid carboxyalkyl ether of ethylene glycol (sodium salt)is titrated to determine the carboxyl content per gram of product.

The concentrations of Solution A, Solution B and Solution C can bevaried and optimized, with the above concentrations as guidelines, toachieve the desired level of carboxyalkylation of the ethylene glycolproduct. For example, varying amounts (˜100 mL to 300 mL) of ˜8 N to 12N sodium hydroxide solutions can be used for Solution A, while theamounts (˜125 mL to 200 mL) and concentration (˜2.75 M to 3.75 M) ofSolution B may also vary as needed, according to amount andconcentration of starting polyether compound.

Example 6 Improved Method of Administering a Derivatized Polyol orPolyether Iron Oxide Complex in a Subject to Provide Minimal DetectableFree Iron

Iron oxide polyol or polyether complexes such as carboxyalkyl-. amino-,amido-, or ester derivatives of reduced dextran iron oxide complexes, orcarboxyalkyl-, amino-, amido- or ester derivatives of ethylene glycoliron oxide complexes in accordance with the present invention areadministered parenterally by bolus injection at a dosage of from about 1mg to about 4 mg of iron/kg of body weight, administered all at once ina 5-mL, 10-mL or up to 15-mL saline solution, wherein administration ofthe complex results in minimal detectable free iron, as measured eitherby atomic absorption spectroscopy as described in Example 2, or asmeasured by a BDI assay as described in Example 3 and the referencestherein.

Example 7 Improved Method of Administering a Polyol or Polyether IronOxide Complex in a Subject to Provide Minimal Anaphylactic Response

Iron oxide polyol or polyether complexes such as carboxyalkyl-. amino-,amido-, or ester derivatives of reduced dextran iron oxide complexes, orcarboxyalkyl-, amino-, amido- or ester derivatives of ethylene glycoliron oxide complexes in accordance with the present invention areadministered parenterally by bolus injection at a dosage of from about 1mg to about 4 mg of iron/kg of body weight, administered all at once ina 5-mL, 10-mL or up to 15-mL saline solution, wherein administration ofthe complex results in minimal anaphylactic response, as measured eitherby an ELISA assay as described in Example 1, or as indicated by volumeof rat paw edema as described in Example 65 and the references therein.

Example 8 Improved Method for Formulating an Iron Oxide Complex with aPolyol or Polyether Having Minimal Dissolution In Vivo

A polyol or polyether iron oxide complex in accordance with the presentinvention is formulated for parenteral administration and supplied in asterile biocompatible liquid solution, preferably sterilized byautoclaving and/or microfiltration with a 0.2 μm filter, at aconcentration of 50, 75, or 100 mg/mL in 1-mL to 5 mL single dose vialsfor intramuscular or intravenous injection. Each mL containsapproximately 0.9% saline solution (sodium chloride) in water, andsodium hydroxide and/or hydrochloric acid may have been used to adjustthe pH to between about 5.2 and 6.5. The formulations of the presentinvention are prepared such that upon administration to a patient theiron oxide complex remains intact in vivo and displays minimaldissolution, as measured by % transferrin saturation, a BDI assay,and/or a bacterial growth assay.

In other methods for administration, the iron oxide is administered as abolus injection wherein the iron oxide is dissolved in a biocompatibleliquid as above, and injected all at once in a high concentration/lowvolume injection. Such injection means a concentration of iron oxide inthe biocompatible liquid at concentrations of between about 50 mg/mL toabout 200 mg/mL, in total volumes from between about 1 mL to about 5 mL.

Example 9 Improved Method for Formulating an Iron Oxide Complex with aPolyol or Polyether that is Immunosilent in a Human Subject

A polyol or polyether iron oxide complex in accordance with the presentinvention is formulated for parenteral administration and supplied in asterile solution, preferably sterilized by autoclaving and/ormicrofiltration with a 0.2 μm filter, at a concentration of about 50,75, 100 or 200 mg/mL in about 1-mL to 5-mL single dose vials forintramuscular or intravenous injection. Each mL contains approximately0.9% saline solution (sodium chloride) in water, and sodium hydroxideand/or hydrochloric acid may have been used to adjust the pH to betweenabout 5.2 and 6.5. The formulations of the present invention areprepared such that upon administration to a patient the iron oxidecomplex presents as an immunosilent agent to the patient, as indicatedby the patient's physical response and confirmed by ELISA assay.

General Procedures for the Synthesis of Reduced Polysaccharides.

Reduced polysaccharides were prepared by treatment with excess sodiumborohydride and generally purified using five cycles of ultrafiltration.Distilled water is used throughout the examples. In the case of thepolysaccharide pullulan, the reduction mixture was used without furtherpurification. In all cases, the products showed less than 5% residualaldehyde content. Residual aldehyde concentration was determined using amodified tetrazolium blue assay (Jue, C. K. et al., J. Biochem. Biophys.Methods, 1985, 11:109-15).

Dextran concentration was determined by a phenol/sulfuric acid assay(Kitchen, R., Proc. Sugar Process. Res. Conf., 1983, 232-47). In caseswhere ultrafiltration was omitted, it was demonstrated that, except forthe dextran T1, the residual borate salts did not affect particleformation. Examples 10 through 13 provide methods of synthesis of reducepolysaccharides T1, T5, and T10 dextrans, and pullulan, respectively.Retention times were determined using a Waters Ultrahydrogel 250 column,SN T52262A33, with 20 mM phosphate buffered saline, 0.4 mL/min flowrate.

Example 10 Reduced Dextran T1

Dextran T1 (10 g) was dissolved in 100 mL water at 25° C., 1.0 g ofsodium borohydride was added, and the mixture was stirred for 12 h. ThepH was brought to 5.0 using 6 M HCl, and 200 mL ethanol (anhydrous) wasadded. The precipitate was collected by centrifugation. Theethanol/water layer was decanted, and the residue was dissolved in 100mL water. Addition of 200 mL of absolute ethanol was used to cause asecond precipitation, and the ethanol/water was again decanted. Theprecipitated product was dissolved in water, and was lyophilized toproduce a white solid, with a 60% yield. The observed HPLC retentiontimes (min) were: for reduced dextran, 24.4; and for native dextran,24.4.

Example 11 Reduced Dextran T5

Dextran T5 (4 g) was dissolved in 25 mL water at 25° C., 83 mg of sodiumborohydride was added, and the mixture was stirred for 12 h. The pH wasbrought to 5.0 using 6 M HCl. The mixture was ultrafiltered against a 1kDa molecular weight cut-off (MWCO) membrane filter. The product waslyophilized to produce a white solid, and a 70% yield was obtained. Theobserved HPLC retention times (min) were: for reduced dextran, 22.9; fornative dextran, 21.9.

Example 12 Reduced Dextran T10

Dextran T10 (5,003 g) was dissolved in 26,011 g water. Sodiumborohydride was added (52.5 g) and the mixture was stirred for 24 hours.The pH was adjusted to 7.1 using 6 N HCl. The product was purified byrepeated ultrafiltration against a 3 kDa ultrafiltration membrane andlyophilized to produce a white solid. Yield: 3129 g. The observed HPLCretention times (min) were: for reduced dextran, 21.6; for nativedextran, 21.1.

Example 13 Reduced Pullulan

Pullulan (90 mg) was dissolved in 0.8 mL water at 25° C., and 1 mg ofsodium borohydride was added. The mixture was stirred for 12 h, and wasused directly in the preparation of USPIO.

General Procedures for Synthesis of a Carboxymethyl Reduced DextranUsing Native Dextran T-10 as a Substrate.

Examples 14-18 describe the synthesis of carboxymethyl reduced dextransfrom native dextran. Two general methods of synthesis are presented, alow dextran concentration method (Example 145) in which the startingconcentration of native dextran was 70 mg/g, and a high dextranconcentration method (Examples 15-18), in which the startingconcentration of native dextran was 240 mg/g.

Example 14 Carboxymethyl Reduced Dextran T10 Prepared by the Low DextranConcentration Method

The following solutions were prepared and cooled to 5° C.: Solution Acontained 4,200 g sodium hydroxide in 10.5 liters of water; and SolutionB contained 2,310 g bromoacetic acid in 5,700 mL water. Solution Ccontained 3,000 g dextran T10 in 7,500 mL water, heated to 38° C.

Sodium hydroxide (600 g) was dissolved in 7.5 liters of water and waswarmed to 38° C. Sodium borohydride (60 g) was added and the mixture wasstirred for 2 min before adding Solution C, followed immediately byadding a second 60 g portion of sodium borohydride. The mixture wasstirred at 38° C. for 30 min, and then cooled to 15° C. Solution A wasadded, keeping the temperature of the solution below 25° C. Solution Bwas added, and the temperature of the solution was maintained below 25°C. The mixture was stirred for 2 hours at room temperature, and wasneutralized to pH 7.5 using 6M HCl cooled to 5° C., maintaining thesolution temperature below 35° C. The mixture was filtered through a 0.2μm filter, and diluted to 80 liters. The product was purified byrepeated ultrafiltration through a 3 kDa MWCO ultrafiltration membrane,again filtered through a 0.2 μm filter and was lyophilized.

The recovered solid, 2,560 g of carboxymethyl reduced dextran T10(sodium salt), showed a carboxyl content of approximately 1,265micromoles carboxyl per gram of product, as determined by titration. Theuse of bromoacetic acid allowed the reaction to proceed at a lowertemperature compared to use of chloroacetic acid, and produced a cleanerproduct as evidenced by its FTIR spectrum (FIG. 2). FIG. 2 shows nocarbonyl absorption other than that of the carboxylate at 1600 cm⁻¹,unlike the FTIR of the product in U.S. Pat. No. 5,204,457 which wasprepared with chloroacetic acid.

Example 15 Carboxymethyl Reduced Dextran CMRD T10 Prepared by the HighDextran Concentration Method

Sodium borohydride (0.4 g) and 0.5 g of a 50% solution weight/weight ofsodium hydroxide in water were added to a solution of 25 g dextran in 50g water. The mixture was stirred 4 hours at room temperature, 19.5 g ofthe 1:1 sodium hydroxide solution and 6.2 g bromoacetic acid were added,and the temperature was kept below 25° C. using an ice bath. The mixturewas then stirred 16 hours at room temperature.

To purify the product, the pH of the mixture was adjusted to pH 6.2using 6 M HCl, and 120 mL ethanol was added. A precipitate formed andwas allowed to settle, and the supernatant was removed by decanting. Theresidue was dissolved in 60 mL water, and 200 mg sodium chloride wasadded, followed by 30 mL ethanol, and the carboxymethyl reduced dextranwas allowed to settle out. The sequence of addition of water and sodiumchloride followed by dissolution of the precipitate and ethanolprecipitation was repeated an additional two times. The residue wasdissolved in 60 mL water, and 1 liter of ethanol was added. Thecarboxymethyl reduced dextran was again allowed to settle out, and thesolid was collected on a medium frit glass filter. The white solid wasdried 24 hours at 50° C. The yield was 27 g of product having 1108micromoles carboxyl per gram as measured by titration (Table 6).

Example 16 Carboxymethyl Reduced Dextran T10 Prepared by the HighDextran Concentration Method

Sodium borohydride (0.4 g) and 0.5 g of 50% sodium hydroxide were addedto a solution of 25 g dextran in 50 g water. The mixture was stirred 4hours at room temperature, 20.0 g 50% of sodium hydroxide and 6.95 g ofbromoacetic acid were added and temperature was kept below 25° C. usingan ice bath while the mixture was stirred for 16 hours at roomtemperature. The product was purified as described in Example 15. Theyield was 23.9 g of product having 1262 micromoles carboxyl per gram asmeasured by titration (Table 6).

Example 17 Carboxymethyl Reduced Dextran T10 Prepared by the HighDextran Concentration Method

Sodium borohydride (0.4 g) and 0.5 g of 50% sodium hydroxide were addedto a solution of 25 g dextran in 50 g water. The mixture was stirred for4 hours at room temperature, and 20.67 g of 50% sodium hydroxide and7.65 g bromoacetic acid were added while the temperature was kept below25° C. using an ice bath. The mixture was stirred for 16 hours at roomtemperature. The product was purified as described in Example 15. Theyield was 24.5 g of product having 1404 micromoles carboxyl per gram asmeasured by titration (Table 6).

Example 18 Carboxymethyl Reduced Dextran CMRD T10 Prepared by the HighDextran Concentration Method

Sodium borohydride (0.4 g) and 0.5 g of 50% solution of sodium hydroxidewere added to a solution of 25 g dextran in 50 g water. The mixture wasstirred for 4 hours at room temperature, and 20.67 g of 50% sodiumhydroxide and 7.65 g of bromoacetic acid were added while thetemperature was kept below 25° C. using an ice bath. The mixture wasstirred for 16 hours at room temperature, and the product was purifiedas described in Example 15. The yield was 23.4 g of product having 1528micromoles carboxyl per gram of product as measured by titration (Table6).

The relationship between amount of bromoacetic acid used in thesynthesis and the resulting incorporation of micromoles of carboxylgroups into dextran was examined using the high dextran concentrationmethod. The relationship was found to be linear (see Table 6 and FIG.4). Reactant masses and carboxymethyl yields for Examples 15 through 18are shown in Table 6.

TABLE 6 Conditions for CMRD synthesis extent and degree ofcarboxymethylation of the product. dextran NaOH, bromoacetic acid,micromoles COOH Example mg/g mg/g mg/g per g product 15 246 96.0 61.01108 16 243 97.2 67.6 1262 17 240 99.2 73.4 1404 18 238 100.3 77.2 1528Synthesis Of Carboxymethyl Reduced Dextran Preparations Using ReducedDextran T-10 by the Low Dextran High Base Method.

Examples 19-23 describe the synthesis of carboxymethyl reduced dextranswith varying degrees of substitution starting with a low concentrationof reduced dextran. In this method, the starting concentration ofreduced dextran was 70 mg/g and the NaOH was at least about 107 mg/g.Table 7 shows that the extent of carboxymethyl substitution increased asthe amount of bromoacetic acid used in the reaction increased.

Example 19 Carboxymethyl Reduced Dextran CMRD T10 Using the Low DextranHigh Base Method

Reduced dextran T10 (15 g) was dissolved in 72 mL water, and 72 mL of 8Msodium hydroxide was added. The mixture was brought to 25° C., and asolution of 1.15 g bromoacetic acid in 3 mL of water was added. Themixture was stirred at room temperature for 1 hour, and then added to a75 mL volume of crushed ice. The pH of the solution was brought to pH6.0 using 6M HCl. After repeated ultrafiltration against a 3 kDaultrafiltration membrane, the product was lyophilized. The yield was13.25 g of product. The recovered solid, carboxymethyl reduced dextranT10 (sodium salt), showed a carboxyl content of approximately 110micromoles carboxyl per gram as determined by titration (Table 7).

Example 20 Carboxymethyl Reduced Dextran T10 Using the Low Dextran HighBase Method

Reduced dextran T10 (150 g) was dissolved in 720 mL water, and 720 mL of8M sodium hydroxide was added. The mixture was brought to 25° C., and asolution of 11.5 g bromoacetic acid in 140 mL water was added. Themixture was stirred at room temperature for 1 hour, added to a 750 mLvolume of crushed ice, and the pH of the solution was brought to pH 6.0with 6M HCl. After repeated ultrafiltration against a 3 kDa MWCOultrafiltration membrane, the product was lyophilized. The yield was126.21 g of recovered solid carboxymethyl reduced dextran T10 (sodiumsalt), having a carboxyl content of approximately 130 micromolescarboxyl per gram product as determined by titration (Table 7).

Example 21 Carboxymethyl Reduced Dextran CMRD T10 Using the Low DextranHigh Base Method

Reduced dextran T10 (150 g) was dissolved in 720 mL water, and 720 mL of8M sodium hydroxide was added. The mixture was brought to 25° C., asolution of 26.6 g bromoacetic acid in 140 mL water was added, and themixture was stirred at room temperature for 1 hour and added to a 750 mLvolume of crushed ice. The pH of the solution was brought to pH 6.0 with6M HCl. After repeated ultrafiltration against a 3 kDa MWCOultrafiltration membrane, the product was lyophilized. The yield was notdetermined. The recovered solid, carboxymethyl reduced dextran T10(sodium salt), showed a carboxyl content of approximately 280 micromolescarboxyl per gram product as determined by titration (Table 7).

Example 22 Carboxymethyl Reduced Dextran CMRD T10 Using the Low DextranHigh Base Method

Reduced dextran T10 (15 g) was dissolved in 72 mL of water, and 72 mL of8M sodium hydroxide was added. The mixture was brought to 25° C., and asolution of 3.45 g of bromoacetic acid in 8 mL water was added. Themixture was stirred at room temperature for 1 hour, and then added to a75 mL volume of crushed ice. The pH of the solution was brought to pH6.0 with 6M HCl. After repeated ultrafiltrations against 3 kDa MWCOultrafiltration membranes, the product was lyophilized. The yield was9.4 g of recovered solid carboxymethyl reduced dextran T10 (sodiumsalt), having a carboxyl content of approximately 450 micromolescarboxyl per gram product as determined by titration (Table 7).

Example 23 Carboxymethyl Reduced Dextran CMRD T10 Using the Low DextranHigh Base Method

Reduced dextran T10 (150 g) was dissolved in 720 mL of water, and 720 mLof 8M sodium hydroxide was added. The mixture was brought to 25° C., anda solution of 58.8 g of bromoacetic acid in 140 mL water was added. Themixture was stirred at room temperature for 1 hour, and was then addedto a 750 mL volume of crushed ice. The pH of the solution was brought topH 6.0 using 6M HCl. After repeated ultrafiltrations against a 3 kDaMWCO ultrafiltration membrane, the product was lyophilized. The yieldwas 127.88 g of the recovered solid carboxymethyl reduced dextran T10(sodium salt), having a carboxyl content of approximately 580 micromolescarboxyl per gram product as determined by titration (Table 7).

Table 7 shows that the extent of carboxymethyl substitution observed wasa function of the amount of bromoacetic acid used in the reaction. Thedata show that generally increasing the amount of bromoacetic acid inthe reaction resulted in increasing levels of COOH in the product. Theyield of carboxymethyl incorporation was also affected by conditionssuch as scale of the reaction, for example, as in Examples 22 and 23.

TABLE 7 Preparation of CMRDs with varying extents of carboxymethylation.dextran NaOH bromoacetic acid micromoles Example mg/g mg/g mg/g COOH/gproduct 19 75 115.7 5.77 110 20 75 115.7 5.77 130 21 73 111.6 16.7 28022 70 107.2 27.3 450 23 70 107.2 27.3 580

Example 24 Carboxymethyl Reduced Dextran T10 from a Commercial Source

Carboxymethyl reduced dextran was purchased from Amersham-Pharmacia. Thesolid showed a carboxyl content of approximately 1887 micromolescarboxyl per gram product as determined by titration.

Examples 25-27 describe synthesis of carboxymethyl dextran from native,non-reduced dextran T-10, T-40, and T-70, respectively.

Example 25 Carboxymethyl Dextran T10

The following solutions were prepared and cooled to 5° C.: Solution A:105.2 g sodium hydroxide in 250 mL water; Solution B: 58.0 g bromoaceticacid in 142.5 mL water; and Solution C, 75.7 g dextran T10 in 187.5 mLwater.

To Solution C and Solution A were added sodium hydroxide (14.4 g)dissolved in 187.5 mL water while maintaining the temperature of thesolution below 25° C. Solution B was added, keeping the temperaturebelow 25° C., and the resulting solution was stirred for 2 hours at roomtemperature, then was neutralized to pH 7.5 with 6M HCl (cooled to 5°C.) while maintaining the solution temperature below 35° C. The mixturewas passed through a 0.2 μm pore size filter, and diluted to 2 liters.The product was purified by repeated ultrafiltration against a 3 kDaMWCO ultrafiltration membrane, 0.2 μm filtered again, and lyophilized.The yield was 53.17 g, and the recovered solid carboxymethyl dextran T10(sodium salt) showed a carboxyl content of approximately 1220 micromolescarboxyl per gram product as determined by titration.

Example 26 Carboxymethyl Dextran T40

The following solutions were prepared and cooled to 5° C.: Solution A:154 g sodium hydroxide in 480 mL water; Solution B: 77 g bromoaceticacid in 260 mL water; and Solution C: 100 g dextran T40 in 400 mL water.

Solution A was added to Solution C all at once. After 5 min, Solution Bwas added and the combined solution was stirred for 120 min while thetemperature was maintained between 20° C. and 25° C. The mixture wasneutralized with 6 M HCl, was 0.2 μm filtered, and diluted to 2 liters.The product was purified by repeated ultrafiltration against 3 kDa MWCOultrafiltration membranes, was 0.2 μm filtered and was lyophilized. Theyield was 105.1 g of recovered solid carboxymethyl dextran T40 (sodiumsalt), which showed a carboxyl content of about 1390 micromoles carboxylper gram product as determined by titration.

Example 27 Carboxymethyl Dextran T70

The following solutions were prepared and cooled to 5° C.: Solution A:154 g sodium hydroxide in 480 mL water; Solution B: 77 g bromoaceticacid 260 mL water; and Solution C: 100 g dextran T70 in 400 mL water.

Solution A was added to Solution C all at once. After 5 min, Solution Bwas added, and the combined solution was stirred, maintaining thetemperature between 20° C. and 25° C. using an ice bath. After 120 min,the solution was neutralized with 6 M HCl. The solution was 0.2 μmfiltered, and diluted to 2 liters. The product was purified by repeatedultrafiltration against 3 kDa MWCO ultrafiltration membranes, was 0.2 μmfiltered again and was lyophilized. The yield was 106.9 g of recoveredsolid carboxymethyl dextran T70 (sodium salt), having a carboxyl contentof about 1380 micromoles carboxyl per gram product as determined bytitration.

General Procedure for the Preparation of Superparamagnetic Colloids forComparison of the Properties of USPIO Preparations Coated with Either ofReduced or Non-Reduced Polysaccharides.

Examples 28-35 were conducted to compare polysaccharide coated ironoxide products obtained from pairs of native and reduced polysaccharidesof identical molecular weights. Identical procedures were utilized forthe preparation of USPIO colloids for each pair of native and reducedpolysaccharide of identical molecular weight. In particular, the samepolysaccharide to iron ratio and iron concentration was used for eachmolecular weight pair. The polysaccharide to iron ratio and ironconcentration utilized for each native and reduced polysaccharide pairwere chosen to yield a 0.2 μm filterable USPIO with a diameter of lessthan 30 nm and a magnetic susceptibility of greater than 20,000×10⁻⁶ cgswith the reduced polysaccharide.

The general procedure involved addition of excess ammonium hydroxide toa solution of iron salts (Fe⁺³/Fe⁺²) and polysaccharide, followed byheating, and performing six cycles of ultrafiltration against waterusing a 100 kDa MWCO membrane filter. After ultrafiltration, the USPIOpreparations formed with reduced polysaccharide were filtered through a0.2 μm filter and stored at 4° C.

It was observed that for iron oxides prepared with a nativepolysaccharide, only the native dextran T10 coated iron oxide wasfilterable through a 0.2 μm filter. The size and magneticsusceptibility, except for those samples containing particulatematerials, were then measured. Particle sizes were determined bymeasurement of dynamic light scattering in a Microtrac® UPA instrument(Honeywell IAC Microtrac, Fort Washington, Pa.) and are reported as themean volume diameter (MVD). Magnetic susceptibility was determined witha Mathey Johnson magnetic susceptibility balance. Iron concentrationswere determined with a bipyridyl assay (Kumar K., J. Liq. Chromatogr.Relat. Technol., 1997, 20, 3351-3364).

Example 28 Preparation of Reduced Dextran T1 Coated USPIO

Reduced dextran T1 (1.7 g) was dissolved in 20 mL water, and a solutionof 3 g of ferric chloride hexahydrate and 1.5 g of ferrous chloridetetrahydrate in 32 g water was added. The mixture was purged withnitrogen for 30 min, cooled to 5° C., and 12.7 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated to 60° C., maintained at this temperature for 40 min, thenincubated at 80° C. for 2 h. The product was subjected to six cycles ofultrafiltration against water using a 100 kDa MWCO membrane filter.After ultrafiltration, the product was filtered through a 0.2 μm filterand stored at 4° C. The product was observed to have the followingproperties: the mean volume diameter (determined by use of a MicrotracParticle Size Analyzer) was 18 nm; the magnetic susceptibility was13,323×10⁻⁶ cgs/g Fe.

Example 29 Preparation of Native Dextran T1 Coated Iron Oxide

Native dextran T1 iron oxide was prepared by the method described abovefor the reduced dextran in Example 28 except that native dextran T1 wasused instead of reduced dextran T1. The product was observed to have thefollowing properties: the mean volume diameter (determined by use of aMicrotrac Particle Size Analyzer) was 2764 nm; the magneticsusceptibility was 1,953×10⁻⁶ cgs/g Fe.

Example 30 Preparation of Reduced Dextran T5 Coated USPIO

Reduced dextran T5 (0.45 g) was dissolved in 13 mL water, and a solutionof 0.5 g of ferric chloride hexahydrate and 0.25 g of ferrous chloridetetrahydrate in 4.5 g water was added. The mixture was purged withnitrogen for 30 min, cooled to 5° C., and 1.42 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated at 80° C. for 2 h, and was purified as described in Example 28.The product was observed to have the following properties: the meanvolume diameter (determined by use of a Microtrac Particle SizeAnalyzer) was 16 nm; the magnetic susceptibility was 33,943×10⁻⁶ cgs/gFe.

Example 31 Preparation of Native Dextran T5 Coated Iron Oxide

Native dextran T5 iron oxide was prepared by the method described abovefor the reduced dextran in Example 30 except that native dextran T5 wasused instead of reduced dextran T5. The product was observed to have thefollowing properties: the mean volume diameter (determined by use of aMicrotrac Particle Size Analyzer) was 1,916 nm.

Example 32 Preparation of Reduced Dextran T10 Coated USPIO

Reduced dextran T10 (2.7 g) was dissolved in 70 mL water, and a solutionof 2.0 g ferric chloride hexahydrate and 1.0 g ferrous chloridetetrahydrate in 27 g water was added. The mixture was purged withnitrogen for 30 min, cooled to 5° C., and 8.5 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated at 80° C. for 2 h and purified as described in Example 28. Theproduct was observed to have the following properties: the mean volumediameter (determined by use of a Microtrac Particle Size Analyzer) was12 nm; the magnetic susceptibility was 31,743×10⁻⁶ cgs/g Fe.

Example 33 Preparation of Native Dextran T10 Coated Iron Oxide

Native dextran T10 iron oxide was prepared by the method described abovefor the reduced dextran in Example 32 except that native dextran T10 wasused instead of reduced dextran T10. The product was observed to havethe following properties: the mean volume diameter (determined by use ofa Microtrac Particle Size Analyzer) was 757 ηm; the magneticsusceptibility was 31,252×10⁻⁶ cgs/g Fe.

Example 34 Preparation of Reduced Pullulan Coated USPIO

Reduced pullulan (0.045 g) was dissolved in 0.4 mL water, and a solutionof 0.106 g ferric chloride hexahydrate and 0.05 g ferrous chloridetetrahydrate in 1.3 g water was added. The mixture was purged withnitrogen for 30 min, cooled to 5° C., and 0.044 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated at 80° C. for 0.67 h and purified as described in Example 28. Theproduct was observed to have the following properties: the mean volumediameter (determined by use of a Microtrac Particle Size Analyzer) was20 nm; the magnetic susceptibility was 27,066×10⁻⁶ cgs/g Fe.

Example 35 Preparation of Native Pullulan Coated Iron Oxide

Native pullulan iron oxide was prepared by the method described abovefor the reduced pullulan in Example 34 except that native pullulan wasused instead of reduced pullulan. The product was observed to have thefollowing properties: the mean volume diameter (determined by use of aMicrotrac Particle Size Analyzer) was 1,184 nm.

Properties of Iron Oxide Preparations Obtained Using Reduced inComparison to Native Polysaccharides (Comparison of Data Obtained fromExamples 28-35).

In general for MRI contrast agents, an iron oxide contrast agentparticle of small size is preferred, for example, a particle having adiameter in the range of 10 to 50 nm. Further, an iron oxide of greatermagnetic susceptibility and of greater homogeneity is preferred.

It is observed from the data of Examples 28-35 that the presence of areduced terminal sugar of a polysaccharide (reduced polysaccharide) usedto coat an iron oxide had an unexpected and substantial effect on thediameter of particles of each of the resulting colloids, compared tosimilarly produced iron oxides made using native non-reducedpolysaccharide. Table 8 shows the size of particles formed for each pairof native and reduced polysaccharides, as indicated by the mean volumediameters (MVD). The concentrations of reduced and nativepolysaccharides were kept constant within each molecular weight group.Concentrations were selected to optimize the synthesis of USPIO withreduced polysaccharide. For all polysaccharides, use of the nativenon-reduced polysaccharide consistently produced a larger particle thandid use of the reduced dextran, so that the reduced polysaccharideconsistently gave the preferred smaller particle.

Further, for each pair of polysaccharides of a given molecular weightthat was synthesized and tested, the USPIO preparation coated withreduced polysaccharides demonstrated a higher magnetic susceptibilityvalue than the corresponding iron oxide preparation synthesized withnative polysaccharide, except for colloids obtained with dextran T10 forwhich magnetic susceptibilities of reduced and native coatings wereequivalent.

These data indicate that use of a reduced polysaccharide in preparationof coated USPIO colloids yields preferred particles of small size,without loss of magnetic susceptibility. The data demonstrate thesurprising effect that reduction of the aldehyde of a polysaccharide hasupon the synthesis of a polysaccharide-coated USPIO.

TABLE 8 Comparison of properties of iron oxides made with native orreduced polysaccharides under conditions that form a USPIO with reducedpolysaccharides. ratio of poly- MVD Exam- poly- saccharide nm MS^(a) plesaccharide per Fe, g/g reduced native reduced native 28, 29 dextran T11.6 18 2,764 13,323  1,953 30, 31 dextran T5 2.9 16 1,916 33,943 b 32,33 dextran T10 4.6 21 757 31,743 31,252 34, 35 pullulan 3.9 20 1,18427,066 b ^(a)Magnetic susceptibility (×10⁻⁶ cgs/g Fe) b The sample wasparticulate, could not be filtered through a 0.2 μm filter, and magneticsusceptibility was not determined.Properties of Iron Oxides Prepared with Native Non-Reduced T1, T5 andT10 Dextrans of Mean Volume Diameter Less than 30 Nm.

Examples 36, 38 and 40 and 37, 39 and 41 show the preparation of ironoxides obtained from native dextran T1, T5, and T10 and from PEG4000,PEG60000, and PEG10000, respectively. Colloids were prepared usingnon-reduced (native) dextrans as described for reduced dextrans(Examples 28, 30, and 32), except that the preparation of these nativenon-reduced dextran particles required about 10- to 34-fold more dextranthan their corresponding reduced dextran counterpart to produce ironoxides of corresponding size. The requirement for increased dextranusage is shown by comparing the dextran to iron ratio of the productsfor corresponding molecular weight pairs of iron oxides shown (Table 9).

The data show that the magnetic properties, and the efficiency ofdextran use during synthesis, of iron oxide particles prepared with eachof native dextrans T1, T5, and T10 were inferior compared withcorresponding properties of particles prepared with each counterpartreduced dextran.

Examples 36 Preparation of Iron Oxide Coated with Native T1 Dextran

A mixture of 0.42 g ferric chloride hexahydrate, 0.21 g ferrous chloridetetrahydrate, and 7.27 g water was filtered through a 0.2 μm filter. A1.0 g portion of this mixture was added to 10 mL of an aqueous solutionof 0.1 g dextran T1/g water. The mixture was purged with nitrogen beforeadding 0.22 mL of 28% ammonium hydroxide solution. The mixture washeated at 80° C. for 1 hour, cooled to room temperature and filteredthrough a 0.2 μm filter. The product was observed to have the followingproperties: the mean volume diameter (determined by use of a MicrotracParticle Size Analyzer) was 27 nm; the magnetic susceptibility was2325×10⁻⁶ cgs/g Fe.

Example 37 Preparation of Iron Oxide Coated with the PolyetherlPolyethylene Glycol 4000 (PEG4000)

A mixture of 0.42 g ferric chloride hexahydrate, 0.21 g ferrous chloridetetrahydrate, and 7.27 g water is filtered through a 0.2 μm filter. A1.0 g portion of this mixture is added to 10 mL of an aqueous solutionof 0.1 g polyethylene glycol 1000/g water (PEG100/g water). The mixtureis purged with nitrogen before adding 0.22 mL of 28% ammonium hydroxidesolution. The mixture is heated at 80° C. for about 1 hour, cooled toroom temperature and filtered through a 0.2 μm filter. The mean volumediameter (determined by use of a Microtrac Particle Size Analyzer) andthe magnetic susceptibility in cgs/g Fe are then determined.

Examples 38 Preparation of Iron Oxide Coated with Native T5 Dextran

Dextran T5 (0.8 g) was dissolved in 9 mL water, and added to 0.63 mL ofa 0.2 μm filtered solution of 51.8 mg ferric chloride hexahydrate and25.9 mg ferrous chloride tetrahydrate in 9.2 mL water. The mixture waspurged with nitrogen before adding 1.4 mL 28% ammonium hydroxidesolution. The mixture was heated at 80° C. for 1 hour, cooled to roomtemperature, and filtered through a 0.2 μm filter. The product wasobserved to have the following properties: the mean volume diameter(determined by use of a Microtrac Particle Size Analyzer) was 20 μm; themagnetic susceptibility was 1285×10⁻⁶ cgs/g Fe

Examples 39 Preparation of Iron Oxide Coated with PEG6000

PEG6000 (0.8 g) is dissolved in 9 mL water, and added to 0.63 mL of a0.2 μm filtered solution of 51.8 mg ferric chloride hexahydrate and 25.9mg ferrous chloride tetrahydrate in 9.2 mL water. The mixture is purgedwith nitrogen before adding 1.4 mL 28% ammonium hydroxide solution. Themixture is heated at 80° C. for about 1 hour, cooled to roomtemperature, and filtered through a 0.2 μm filter. The mean volumediameter (determined by use of a Microtrac Particle Size Analyzer) andthe magnetic susceptibility in cgs/g Fe are then determined.

Example 40 Preparation of Iron Oxide Coated with Native T10 Dextran

Dextran T10 (9420 g) was dissolved in 14915 g water. A 14915 g portionof this mixture was filtered through a 0.2 μm filter, and added to thereaction vessel. Ferric chloride hexahydrate (891 g) was dissolved in713 g water. A 1129 g portion was 0.2 μm filtered and added to thereaction vessel containing the dextran. The mixture was cooled to 5° C.with stirring overnight while bubbling nitrogen through the mixture.Before the last 30 min. of the nitrogen purge, a 580 g portion of a 0.2μm filtered solution of 359 g ferrous chloride tetrahydrate in 477 gwater was added. To this mixture was added 786 g of 28% ammoniumhydroxide solution, cooled to 5° C. The mixture was heated to 80° C.,incubated at 80° C. for 2 hours, and then poured into 80 liters of waterheated to 80° C. The mixture was allowed to cool overnight, 0.2 μmfiltered, and purified by ultrafiltration using a 100 kDaultrafiltration membrane. The product was 0.2 μm filtered. The productwas observed to have the following properties: the mean volume diameter(determined by use of a Microtrac Particle Size Analyzer) was 21 nm; themagnetic susceptibility was 32,712×10⁻⁶ cgs/g Fe.

TABLE 9 Magnetic susceptibility and particle size properties ofpolysaccharide coated iron oxides: a comparison of native dextrans(Examples 36-40) with respective reduced dextrans (Examples 28, 30 and32) under conditions to give particles of less than 30 nm MVD withmaximum magnetic susceptibility. iron oxides prepared with nativedextran Example dextran type dextran/Fe (g/g)^(b) MVD (nm) MS^(a) 36dextran T1 55 27 2,325 28 dextran T5 44 20 1,285 40 dextran T10 44 2132,712 28 dextran T1 1.6 18 13,323 30 dextran T5 2.9 16 33,943 32dextran T10 4.6 12 31,743 ^(a)Magnetic susceptibility (×10⁻⁶ cgs/g Fe)^(b)The polysaccharide/Fe ratio was varied for each dextran in order toobtain a USPIO with a MVD of less than or equal to 30 nm.

Example 41 Preparation of Iron Oxide Coated with PEG10000

PEG10000 (9000 g) is dissolved in 14000 g water. A 14000-g portion ofthis mixture is filtered through a 0.2 μm filter, and added to thereaction vessel. Ferric chloride hexahydrate (890 g) is dissolved in 710g water. A 1130-g portion is 0.2 μm filtered and added to the reactionvessel containing the PEG10000. The mixture is cooled to 5° C. withstirring overnight while bubbling nitrogen through the mixture. Beforethe last 30 min. of the nitrogen purge, a 580 g portion of a 0.2μm-filtered solution of 360 g ferrous chloride tetrahydrate in 480 gwater is added. To this mixture is added 790 g of a 28% ammoniumhydroxide solution, and the mixture is then cooled to 5° C. The mixtureis heated to 80° C., incubated at 80° C. for 2 hours, and then pouredinto 80 liters of water heated to 80° C. The mixture is allowed to coolovernight, 0.2 μm filtered, and purified by ultrafiltration using a100-kDa ultrafiltration membrane. The product is then 0.2 μm filtered.The mean volume diameter (determined by use of a Microtrac Particle SizeAnalyzer) and the magnetic susceptibility in cgs/g Fe are thendetermined.

Preparation USPIOs Coated with Carboxymethyl Native Dextran T10 andCarboxymethyl Reduced Dextran T10 Containing Varying Degrees ofCarboxymethylation.

Examples 42 and 43 describe preparation of USPIO coated withcarboxymethyl native and reduced dextran T10, respectively. Examples44-48 describe the synthesis of USPIO compositions coated withcarboxymethyl reduced dextran T10 preparations, containing varyingdegrees of carboxymethylation. Examples 49-53 describe the solubility ofpreparations containing ferric chloride and carboxymethyl reduceddextran T10 containing varying degrees of carboxymethylation.

Example 42 Preparation of USPIO Coated with Carboxymethyl Dextran T10

Carboxymethyl dextran T10 (60 g, prepared by the method Example 25) wasdissolved in 532 g water. A solution of 14.7 g ferric chloridehexahydrate, 7.2 g ferrous chloride tetrahydrate, and 100 mL water, wasfiltered through a 0.2 μm, and added. The mixture was cooled to 10° C.,purged with nitrogen, and 52.2 mL of 28% ammonium hydroxide solution wasadded with stirring. The mixture was heated to 75° C., maintained at 75°C. for 30 min, diluted with 2.5 liter water, and filtered through a 0.2μm filter. The product was purified by repeated ultrafiltration againsta 100 kDa MWCO membrane, concentrated to 20 mg Fe/mL, and again filteredthrough a 0.2 μm filter. The product was observed to have the followingproperties: MVD (determined by use of a Microtrac Particle SizeAnalyzer) was 19 nm; the magnetic susceptibility was 27,835×10⁻⁶ cgs/gFe; and the carboxyl content was 1,220 micromoles per gram of the CMRD.To determine stability in response to autoclaving, a sample of theproduct was placed in a sealed 5 mL glass vial, and heated to 121° C.for 30 min (see Table 13).

Example 43 Preparation of USPIO Coated with Carboxymethyl ReducedDextran T10

Reduced carboxymethyl dextran T10 (40 g prepared in Example 14) wasdissolved in 1,038 mL water and was filtered through a 0.2 μm pore sizefilter. A 0.2 μm filtered solution of 30 g ferric chloride hexahydrateand 15 g of ferrous chloride tetrahydrate in 374 mL of water was addedto the dextran, with a 31 mL water wash. The solution was cooled to 10°C., and 114 g of 28% ammonium hydroxide was added. The colloidal mixturewas heated to 78° C. and maintained at that temperature for one hour.The solution was then diluted to 3 liter with water, cooled to 10° C.,and ultrafiltered 6 times with a YM-100 filter membrane (100 kDa MWCO).A final concentration of 21.1 mg Fe/g was obtained. The product wasobserved to have the following properties: the mean volume diameter(Microtrac Particle Size Analyzer) was 21 nm; the magneticsusceptibility was 32,732×10⁻⁶ cgs/g Fe; and the carboxyl content was1,265 micromoles per gram of the CMRD. The content of the particle wasdetermined to be about 50% Fe and 50% dextran. To determine stability inresponse to autoclaving, a sample of the product was placed in a sealed5 mL glass vial, and heated to 121° C. for 30 min (see Table 13).

Example 44 Preparation of USPIO Coated with Carboxymethyl ReducedDextran T10 Having 110 Micromoles Carboxyl Per Gram

Carboxymethyl reduced dextran T10 (4 g, prepared in Example 19) wasdissolved in 85 mL water. To this was added a 0.2 μm filtered mixture of2.99 g ferric chloride hexahydrate, 1.49 g ferrous chloridetetrahydrate, and 37.3 mL water. The mixture was cooled to 10° C.,purged with nitrogen, 11.4 g of 28% ammonium hydroxide solution wasadded with stirring the mixture was heated to 90° C., maintained at 78°C. for 60 minutes, and then maintained at 78° C. while bubbling airthrough the mixture. The mixture was diluted with 1.5 liters of water,and was filtered through a 0.2 μm filter. The product was purified byrepeated ultrafiltration against a 100 kDa MWCO membrane and againfiltered through a 0.2 μm filter.

Example 45 Preparation of USPIO Coated with Carboxymethyl ReducedDextran T10 Having 130 Micromoles Carboxyl Per Gram

Carboxymethyl reduced dextran T10 (40 g, prepared in Example 20) wasdissolved in 850 mL water. To this was added a 0.2 μm filtered mixtureof 29.9 g ferric chloride hexahydrate, 14.9 g ferrous chloridetetrahydrate, and 373 mL water. The mixture was cooled to 10° C., purgedwith nitrogen, 114 mL of 28% ammonium hydroxide solution was added withstirring, the mixture was heated to 90° C., maintained at 78° C. for 60min, and then maintained at 78° C. while bubbling air through themixture. The mixture was diluted with 1.5 liters of water, and filteredthrough a 0.2 μm filter. The product was purified by repeatedultrafiltration against a 100 kDa MWCO membrane, concentrated to 20 mgFe/mL, and again filtered through a 0.2 μm filter.

Example 46 Preparation of USPIO Coated with Carboxymethyl ReducedDextran T10 Having 280 Micromoles Carboxyl Per Gram

Carboxymethyl reduced dextran T10 (4 g, prepared in Example 21) wasdissolved in 85 mL water. To this was added a 0.2 μm filtered mixture of2.99 g ferric chloride hexahydrate, 1.49 g ferrous chloridetetrahydrate, and 37.3 mL water. The mixture was cooled to 10° C., andpurged with nitrogen. To the mixture was added with stirring 11.4 g of28% ammonium hydroxide solution, the mixture was heated to 90° C.,maintained at 78° C. for 60 min, and then maintained at 78° C. while airwas bubbled through the mixture. The mixture was diluted with 1.5 litersof water, and filtered through a 0.2 μm filter. The product was purifiedby repeated ultrafiltration against a 100 kDa MWCO membrane, followed byfiltration through a 0.2 μm filter.

Example 47 Preparation of USPIO Coated with Carboxymethyl ReducedDextran T10 Having 450 Micromoles Carboxyl Per Gram

Carboxymethyl reduced dextran T10 (4 g, prepared in Example 22) wasdissolved in 85 mL water. To this was added a 0.2 μm filtered solutionof 2.99 g ferric chloride hexahydrate, 1.49 g ferrous chloridetetrahydrate, and 37.3 mL water. The mixture was cooled to 10° C., andpurged with nitrogen before adding 11.4 g of 28% ammonium hydroxidesolution with stirring. The mixture was heated to 90° C., maintained at78° C. for 60 min, and then maintained at 78° C. while air was bubbledthrough the mixture. The mixture was diluted with 1.5 liters of water,filtered through a 0.2 μm filter, and was purified by repeatedultrafiltration against a 100 kDa MWCO membrane followed by filtrationthrough a 0.2 μm filter.

Example 48 Preparation of USPIO Coated with Carboxymethyl ReducedDextran T10 Having 580 Micromoles Carboxyl Per Gram

Carboxymethyl reduced dextran T10 (40 g, prepared in Example 23) wasdissolved in 85 mL water. To this was added a 0.2 μm filtered solutionof 29.9 g ferric chloride hexahydrate, 14.9 g ferrous chloridetetrahydrate, and 373 mL water. The mixture was cooled to 10° C., purgedwith nitrogen, 11.4 g of 28% ammonium hydroxide solution with stirring.The mixture was heated to 90° C., maintained at 78° C. for 60 min, thenmaintained at 78° C. while bubbling air through the mixture. The mixturewas diluted with 1.5 liters of water and filtered through a 0.2 μmfilter, and was purified by repeated ultra-filtration against a 100 kDaMWCO membrane followed by filtration through a 0.2 μm filter.

The effect of degree of carboxymethylation of the CMRD coated USPIOs oncolloid size was compared. Examples 43-48, Table 10. The MVD values ofthe resulting colloids were reasonably uniform between CMRD preparationscontaining 110 to 1265 micromoles of carboxyl per gram of product.

TABLE 10 Particle sizes of USPIO colloids prepared with dextran T10CMRDs having varying degrees of carboxymethylation. Example # micromolesCOOH/g dextran mean volume diameter, nm 44 110 12 45 130 15 46 280 18 47450 16 48 580 20 49 1265 21

Example 49 Mixing of Carboxymethyl Reduced Dextran T10 Having 1,108Micromoles Carboxyl Per Gram with Ferric Chloride Solution

As a step in particle synthesis, ferric chloride (0.3 g) was dissolvedin 15 mL water and was filtered through a 0.2 μm pore size filter.Carboxymethyl reduced dextran (prepared in Example 15) was added, themixture was shaken, and was cooled to 10° C. No precipitate wasobserved.

Example 50 Mixing of Carboxymethyl Reduced Dextran T10 Having 1,262Micromoles Carboxyl Per Gram with Ferric Chloride Solution

Ferric chloride (0.3 g) was dissolved in 15 mL water and was filteredthrough a 0.2 μm pore size filter. Carboxymethyl reduced dextran(prepared in Example 16) was added, the mixture was shaken, and wascooled to 10° C. No precipitate was observed.

Example 51 Mixing of Carboxymethyl Reduced Dextran T10 Having 1,404Micromoles Carboxyl Per Gram with Ferric Chloride Solution

Ferric chloride (0.3 g) was dissolved in 15 mL water and was filteredthrough a 0.2 μm pore size filter. Carboxymethyl reduced dextran(prepared in Example 17) was added, the mixture was shaken, and wascooled to 10° C. No precipitate was observed.

Example 52 Mixing of Carboxymethyl Reduced Dextran T10 Having 1,528Micromoles Carboxyl Per Gram with Ferric Chloride Solution

Ferric chloride (0.3 g) was dissolved in 15 mL water and was filteredthrough a 0.2 μm pore size filter. Carboxymethyl reduced dextran(prepared in Example 18) was added, the mixture was shaken, and wascooled to 5° C. An orange white precipitate was observed.

Example 53 Mixing of Carboxymethyl Reduced Dextran T10 Having 1,887Micromoles Carboxyl Per Gram with Ferric Chloride

Ferric chloride hexahydrate (30.3 g) and ferrous chloride (14.8 g) weredissolved in 402.9 mL water and filtered through a 0.2 μm pore sizefilter. Carboxymethyl reduced dextran T10 (40.3 g in 1,033 ml, preparedin Example 24) was added, the mixture was shaken, and was cooled to 5°C. An orange white precipitate was observed.

The effect of varying the degree of carboxymethylation of CMRDs on thefirst step of the CMRD-USPIO synthesis, i.e., combining the aqueousmixtures of CMRD with the iron chloride solutions, was analyzed. Thevarious CMRD preparations were mixed with iron salts at a fixed ironconcentration, the CMRD preparations differing only in degree ofcarboxymethylation as described in Examples 49-53. From 1,108 to 1,404micromoles carboxyl per gram dextran, the CMRD formed a homogeneousmixture in the presence of ferric chloride (Table 11).

TABLE 7 Precipitation of CMRDs having varying levels of carboxyl groupsafter addition of iron salts from mixtures of CMRD (25 mg/g solution)and ferric chloride (19 mg/g solution). Example # micromoles COOH/gdextran precipitate 49 1,108 no 50 1,262 no 43 1,265 no 51 1,404 no 521,528 yes, at 5° C. 53 1,887 yes, at 25° C.

At greater than 1,404 micromoles carboxyl per gram dextran, addition offerric chloride under the conditions and concentrations of the USPIOsynthesis to the CMRD solution produced an orange white precipitate.Even at higher temperatures, where many compounds can be soluble, theprecipitates persisted. The data in Table 11 shows that there is anupper level in modification of CMRD that can be used in the preferredmethod of CMRD-USPIO synthesis.

Example 54 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Preparation of a Magnetic Sol

To prepare a magnetic sol, 60 g of 28% of ammonium hydroxide at 25° C.was added to a solution having 30.0 g ferric chloride hexahydrate and15.1 g ferrous chloride tetrahydrate in 321 g of water. After 5 minutesof mixing, sufficient concentrated HCl was added to obtain a pH of 1.6.The sol was ultrafiltered with a 100 kDa MWCO membrane filter to achievea pH of 3.25, using water as diluent. The magnetic sol was passedthrough a filter of pore size 0.2 μm, then concentrated to 50 mg Fe/g,and stored at 5° C. The yield of iron was 55%, and the product wasobserved to have an MVD of 16 nm.

Example 55 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Preparation of a Non-Magnetic Sol

To a solution of 2.9 g of ferric chloride hexahydrate in 30 mL of waterwas added 10 mL of 10 M NaOH. The mixture was stirred for 5 min, dilutedto 200 mL with water, and the product was collected by filtration. Theresidue was again mixed with water and filtered. The residue was addedto 40 mL water and the pH was adjusted to 2.0. The product was observedto have an MVD of 10 nm.

Example 56 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Coating of a Magnetic Sol withReduced Dextran T10

Reduced dextran T10 (60 mg; Example 12) was dissolved in 1.74 mL waterand combined with 0.24 mL of magnetic sol (13 mg Fe) prepared accordingto Example 54.

The mixture was incubated for 15 min, and the pH was adjusted to 7.4with sodium hydroxide. The particle size (MVD) was determined to be 85nm.

Example 57 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Coating of a Magnetic Sol withNative Dextran T10

Native dextran T10 (60.8 mg) was dissolved in 1.74 mL water, andcombined with 0.24 mL of magnetic sol (13 mg Fe) prepared according toExample 54. The mixture was incubated for 15 min and the pH was adjustedto 7.4 with sodium hydroxide. The particle size (MVD) was determined tobe 1,973 nm.

Example 58 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Coating of a Magnetic Sol withCMRD T10

75 mg of CMRD T10 (Example 14) dissolved in 1.34 mL water was added to0.66 mL of magnetic sol (33 mg Fe) prepared according to Example 54. Themixture was incubated for 15 min at 37° C., and the pH was adjusted to7.95 (plus or minus 0.4) with sodium hydroxide. The mixture wasconcentrated using a 300 kDa ultrafiltration filter. The product wasobserved to have an MVD of 41 nm.

Example 59 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Adjusting the pH of the MagneticSol to 7.4

A magnetic sol as prepared in Example 54 was adjusted to a pH of 7.4. Aprecipitate was observed.

Example 60 Synthesis of Iron Oxide Sols and their Stabilization withNative and Reduced Dextrans and CMRD: Coating of a Non-Magnetic Sol withCMRD T10

A non-magnetic sol prepared according to Example 55 (35 ml) was addeddrop-wise to 35 mL of a 50 mg/g aqueous solution of CMRD T10 preparedaccording to Example 14. The pH was adjusted to 7.0 with 1 N NaOH, thesolution was heated to boiling, cooled to room temperature, and wascentrifuged at 6,000 rpm for 20 min. The supernatant was passed througha filter having a 0.2 μm pore size, and autoclaved at 121° C. for 30min. The product was observed to have a MVD of 86 nm.

Examples 54-60 show that in the absence of a dextran, or in the presenceof a native dextran, a gross iron precipitate forms. Only reduceddextran and CMRD yielded a magnetic sol as a stable colloid.

Example 61 Preparation of CMRD Coated Non-Magnetic Iron Oxide ColloidUsing Base Co-Precipitation of Ferric Chloride and CMRD

Carboxymethyl reduced dextran T10 (19.2 g) (Example 14) was dissolved in300 g water, was filtered through a 0.2 μm filter, and an additional160.8 g of water was added. This solution was added to 120 mL of 0.2 μmfiltered aqueous 0.3 M ferric chloridehexahydrate. To this mixture wasadded 32 mL of aqueous 6N sodium hydroxide. The mixture was heated to100° C. for 3 hours, cooled to room temperature, and ultrafiltered to afinal volume of 50 mL. The product was observed to have an MVD of 30 nm.A portion of this material was placed in a bottle under nitrogen for 30min at 121° C. The autoclaved product had an MVD of 69 nm.

Example 62 Effect of Autoclaving on Reduced and Native Dextran Colloids:Stability to Autoclaving of USPIOs Coated with Native Dextran andReduced Dextran and CMRD

Colloid preparations, each at a concentration of 20 mg Fe/g, wereautoclaved for 30 min at 121° C. Following autoclaving, measurementswere made of bound dextran that was calculated as the difference betweentotal and free dextran, using a phenol/sulfuric acid assay. Free dextranwas separated from the colloid by ultrafiltration. Table 12 shows thatcolloid preparations having USPIOs coated with a reduced dextran havegreater stability than USPIOs coated with a native dextran. The reduceddextran coated USPIO maintained its small size following autoclaving, asthe MVD of the post autoclaved material was increased only 1.3-foldcompared to the MVD of the pre autoclaved material. In contrast, USPIOcoated with native dextran increased in size 28-fold followingautoclaving. The data show that following autoclaving, reduced dextranremains more tightly bound to the iron particle compared to nativedextran.

A second type of increased stability achieved herein by use of reduceddextran to coat USPIO is the property of pH of the bulk solvent. The pHof USPIO coated with reduced dextran dropped 0.9 pH units followingautoclaving, compared to a drop of 1.6 pH units for USPIO coated withnative dextran.

Even greater stability to the autoclaving process was observed forparticles coated with carboxymethyl reduced dextran compared tocarboxymethyl native dextran. The data in Table 13 indicate that USPIOcoated with carboxymethyl non-reduced native dextran showed a 10-50 foldincrease in amount of particulate matter following autoclaving. Incontrast, USPIO coated with carboxymethyl reduced dextran experienced nochange in size or quantity of particulate matter upon autoclaving.Another indication of the stabilizing effect that the carboxymethylreduced polysaccharide coatings confer on the colloid suspension andbulk solvent was the stability of the solvent pH. The data in bothTables 12 and 13 show that the particles coated with reduced dextran hadsignificantly improved pH stability upon autoclaving, compared to thosecoated with native dextran.

TABLE 12 Effect of autoclaving on pH, size, and bound polysaccharide ofcolloids coated with native and reduced dextran. pre autoclaved postautoclaved^(a) bound bound dextran dextran MVD dextran MVD Examplecoating H g/g nm H g/g nm 40 native T10 .0 0.79 21 .5 0.56 587 32reduced T10 .4 1.26 18 .7 0.96 23 ^(a)Samples were prepared at aconcentration of 20 mg iron per mL and autoclaved for 30 minutes at 121°C.

TABLE 13 Effect of autoclaving on pH, size, and particulates of colloidscoated with carboxymethylated reduced and carboxymethylated nativenon-reduced dextran. particulates^(a) >10 microns >25 microns MVD pHnumber/mL number/mL dextran autoclaved Example coating pre pre pre prepre pre pre post 42 CMD^(b) 9 8 .5 .8 5 33 240 43 CMRD^(c) 5 8 .0 .9 5^(a)Particulates were determined by USP analysis. ^(b)CMD, carboxymethyldextran (native) ^(c)CMRD, carboxymethyl reduced dextran ^(d)Sampleswere prepared at a concentration of 20 mg iron per mL and autoclaved for30 minutes at 121° C.

Example 63 Procedures for Determining Relaxation Properties of VariousContrast Agents

Nuclear magnetic (NM) measurements (0.47T) were obtained in a BrukerInstruments pc120 table-top NM sample analyzer operating at 20 MHZ(Proton). Half a milliliter of each sample was placed in the 10 mm NMtubes for relaxivity measurements on the minispec. The placement of thesample in the sample chamber was optimized. The standards were run andtheir values recorded in the log.

Standard procedures were used for T1 and T2 determinations, and theirvalues were recorded. T1 was measured using an inversion recoverytechnique. According to the IR technique, the sample is exposed to a180° pulse and then a 90° pulse to put the magnetization in the plane ofdetection. After sampling, the time between the 180 and 90-degree pulsesis changed, and sampled again. This is done for several durations. Theresulting signals are governed by the equation [M_(∞)−M(t)]/M_(∞)=(1−cosθ)exp(−t/T1). When a 3 parameter fit to data is performed, M_(∞), θ, andT1 are calculated.

T2 was measured using the CPMG technique, where a linear train of 180°pulses of variable length is provided to the sample. The amplitude ofevery second echo is measured. A fit is performed on the accumulateddata using a two parameter (M_(o) and T2) fit. WhereM(t)=M_(o)exp(−t/T2), a plot of ln(M(t)) versus t is linear with a slopeof −1/T2. The inverse of the T1 and T2 was graphed with respect to theiron concentration of the sample. From the slope of best fit line therelaxivity was determined.

TABLE 14 Relaxivity Suscep- Material Coating tibility MW (kDa) R1 R2R2/R1 Example 43 reduced 38,200 10 35.3 64.8 1.8 carboxy- methyl dextranCombidex ® Dextran- 28,000 9.6 21.7 60.3 2.8 T10 Gd-DTPA 172 4.5 5.7 1.3

Example 64 Toxicity of Reduced Dextran, Non-Reduced Dextran, and CMRDCoated Colloids in Rats

An anaphylactic shock type of reaction to dextran can be exhibited byrats and by a small but significant fraction of the human population(Squire, J. R. et al., “Dextran, Its Properties and Use in Medicine,”Charles C. Thomas, Springfield, Ill., 1955). The reaction resemblesanaphylactic shock but does not require prior sensitization, and ischaracterized in rats by the rapid development of prostration, diffuseperipheral vasodilation, and edema of paws, snout and tongue (Voorhees,A. B. et al., Proc. Soc. Exp. Biol. Med. 1951, 76:254). When accompaniedby barbiturate anesthesia, it produces marked hypotension and cyanosis(Hanna, C. H. et al., Am. J. Physiol. 1957, 191:615).

A procedure to measure the extent of rat paw edema response was employedto determine if the presence of reduced dextrans or their derivatives,rather than non-reduced native dextrans, in the coating of the ironoxide colloids could decrease or eliminate potential human adversereactions upon intravenous injection. Rat paw edema was measured as thevolume of the paw prior to and subsequent to injection of test material,using a plethysmometer, which is a differential volume measuring device.The dose of test material was injected, and a second reading was takenafter a designated interval, and the percent change in paw volume wascalculated. The dose administered in these studies was 100 mg Fe/kg bodyweight, a dose much greater than that used as an imaging agent in rats,pigs, and humans (see Examples 67-70).

The results observed following administration of iron oxides coated witheach of reduced and non-reduced T10 dextrans are shown in Table 15. Amarked decrease in the edematous anaphylactic response was observed inthose rats which were administered a USPIO preparation having thereduced dextran or reduced dextran derivatives as a coating, compared tothose rats administered a USPIO preparation having a native non-reduceddextran coating.

TABLE 15 Effect of native and reduced polysaccharide coated particles onrat edema. % Example coating and particle edema 40 native dextran coatedUSPIO >50 32 reduced dextran coated USPIO 13 42 carboxymethyl nativedextran coated USPIO 39 60 carboxymethyl reduced dextran non-magnetic 12colloid 43 carboxymethyl reduced dextran coated USPIO 0

The effect of the CMRD-USPIO preparations having increasing levels ofcarboxymethyl substitution on the extent of anaphylactic response,measured as percent edema, is shown in Table 16. The data show that athreshold level of substitution was necessary to reduce the edematousresponse, and that once this threshold of substitution was achieved, thedecrease in response of the rats to dextran was a surprising eliminationof the edematous response. That is, no edema was observed at 1,265micromoles of carboxyl per gram.

TABLE 16 Extent of rat paw edema as a function of amount ofcarboxymethylation of dextran coating of USPIOs. micromol COOH Exampleper g dextran % edema 44 110 24 45 130 54 46 280 81 47 450 37 48 580 10543 1,265 0

Example 65 Toxicity of Iron Oxide Complexes with a Polyol, Polyether, orCarboxyalkylated Polyol or Polyether in Rats

An anaphylaxis type of reaction to dextran has been exhibited by ratsand by a small but significant fraction of the human population (Squire,J. R. et al., “Dextran, Its Properties and Use in Medicine,” Charles C.Thomas, Springfield, Ill., 1955). The reaction resembles anaphylacticshock but does not require prior sensitization, and is characterized inrats by the rapid development of prostration, diffuse peripheralvasodilation, and edema of paws, snout and tongue (Voorhees, A. B. etal., Proc. Soc. Exp. Biol. Med. 1951, 76:254). When accompanied bybarbiturate anesthesia, it produces marked hypotension and cyanosis(Hanna, C. H. et al., Am. J. Physiol. 1957, 191:615).

A modified procedure as described in Example 64 is used to measure theextent of rat paw edema response, as an indicator of whether thepresence of iron oxide polyol or polyether complexes, or iron oxidecomplexes with carboxyalkyl-, amino-, amido-, ester-, derivatives ofpolyols or polyethers, will cause potential human adverse reactions uponintravenous injection. Rat paw edema is measured as the volume of thepaw prior to and subsequent to injection of test material, using aplethysmometer, which is a differential volume measuring device. Thedose of test material is injected, and a second reading is taken after adesignated interval, and the percent change in paw volume is calculated.The dose administered in these studies can be up to 100 mg Fe/kg bodyweight, a dose much greater than that used as an imaging agent orhematinic agent in rats, pigs, and humans (see Examples 67-70) andcomparable to recommended dosages for Ferrlecit® and Venofer® when usedas hematinic agents (see p. 21 line 17 through p. 22, line 2, above).

For embodiments in accordance with the present invention, minimal pawedema is observed with the iron oxide polyol or polyether complexes ofthe present invention, as measured by the volume of the paw prior to andafter parenteral administration of the complexes. This is in markedcontrast to rats administered other known polyol or polyether iron oxidecomplexes such as Ferrlecit® or Venofer®.

Example 66 Guinea Pig Reaction Test

Similarly, a guinea pig anaphylaxis reaction test is also used as astandard test model for measuring the likelihood of anaphylacticresponse in humans, and is a test required by the FDA for almost allparenterally administered new compounds. In this test, guinea pigs areinjected with small amounts of the test compound beneath the skin for aperiod of between about 6 to 8 weeks, to cause the guinea pigs to buildup antibodies to the test compound and to be sensitized to the testcompound. Then, a full dose of the test compound is administered to theguinea pig by parenteral administration. The full dose administered inthese studies can be up to 100 mg Fe/kg body weight, a dose much greaterthan that used as an imaging agent or hematinic agent in rats, pigs, andhumans (see Examples 67-70) and comparable to recommended dosages forFerrlecit® and Venofer® when used as hematinic agents. For thepre-sensitization injections, the small amount of polyol or polyetheriron oxide complex administered beneath the skin for 6 to 8 weeks can befrom about 0.1 mg/kg body weight or less, to about 0.5-1 mg/kg bodyweight, depending on the individual test compound administered. If ananaphylactic response is observed, there is a strong presumption thatthe test compound will cause anaphylaxis in human subjects.

For embodiments in accordance with the present invention, minimal guineapig anaphylaxis is observed with the iron oxide polyol or polyethercomplexes of the present invention, as measured by the incidence ofguinea pig anaphylaxis to the polyol or polyether iron oxide complexesafter parenteral administration following pre-sensitization. This is inmarked contrast to guinea pigs administered other known polyol orpolyether iron oxide complexes such as Ferrlecit® or Venofer®.

Example 67 Toxicity Studies in Rats of Reduced and Non-Reduced Dextrans

The procedure used in Example 64 was used to determine if the coatingalone, that is, reduced dextrans or their derivatives rather thannon-reduced native dextrans, could eliminate potential human adversereactions upon intravenous injection. Rat paw edema was measured as thevolume of the paw prior and subsequent to injection, as in Example 64.The dose administered in these studies was, as above, 100 mg testsubstance/kg body weight.

The results observed following administration of reduced and non-reducedT10 dextrans were similar for each material (Table 17). Reduced dextranT10 elicited the same extent of edema as native dextran T10. Eliminationor decrease in edema could not be attributed merely to reduction of thedextran.

TABLE 17 Effect of native and reduced 10 kDa polysaccharides on ratedema showing mere reduction has no significant effect. Example testdextran % edema Dextran T-10 native T10 61 (commercial^(a)) 12 reducedT10 67 ^(a)Obtained from Pharmacia-Upjohn (Piscataway, NJ)

Table 18 shows the effect of increased levels of carboxymethylsubstitution of reduced dextran on the extent of anaphylactoid response,measured as percent edema. The data show that above a threshold level ofcarboxymethyl substitution, edema was decreased or eliminated. Fordextrans above this threshold level of substitution, the decrease in thetoxic response of the rats to dextran was a surprising elimination ofresponse, that is, no edema was observed.

TABLE 18 Relationship between rat paw edema and degree ofcarboxymethylation of dextran T10 preparations. micromol COOH/ Exampletest substance g per dextran % edema 19 carboxymethyl reduced 110 65 20carboxymethyl reduced 280 60 22 carboxymethyl reduced 450 56 14carboxymethyl reduced 1,265 6 24 carboxymethyl reduced 1,887 1 25carboxymethyl native 1,220 0

Example 68 Pharmacokinetics of CMRD Coated USPIO in the Rat: BloodClearance

Three male CDA rats (Charles River Laboratories, Wilmington, Mass.;weight range 272 to 290 g) were anaesthetized intraperitoneally with along lasting anesthetic, Inactin (100 mg per kg body weight). Thefemoral artery and vein were exposed by a small incision at thehip-femur joint, and the artery was cannulated with PESO tubingconnected to a 1 mL syringe filled with heparinized saline (10 units perml). To serve as a baseline, 0.25 mL of arterial blood was collected attime zero, and CMRD coated USPIO (Example 43) was injected into thefemoral artery. Blood samples of 0.25 mL were collected at the timesindicated in FIGS. 5 and 6.

T2 magnetic relaxation times were measured in each sample, and therelaxivity (1/T2) was calculated. First-order reaction kinetics wereused to determine the half-life of the sample in the blood (t_(1/2)).The equation used to fit the data was:1/T2−1/T _(baseline) =Ae ^(−kt)where 1/T₂ is the relaxivity of the blood at time t post-injection;1/T_(baseline) is the baseline relaxivity, and A e^(−kt) represents thefirst-order decay of the test material from the blood. Taking thenatural log of each side of this equation yields:ln(1/T2−1/T _(baseline))=−kt+ln A ₀

According to this second equation, a graph of ln(1/T2−1/T_(baseline))versus time, t, should give a straight line with slope −k (the firstorder rate constant) and intercept ln A₀ (which equals ln(1/T2−1/T_(baseline) at time zero) if the rate of removal of the USPIOfrom blood follows first order kinetics. FIG. 6 shows that a straightline was obtained. The half-life (t_(1/2)), which is the time that theamount of CMRD coated USPIO decreased to one half its amount ofconcentration in the blood, was determined to be 67 min, with a range of61 to 75 min at a confidence level of 95%.

Example 69 Magnetic Resonance Imaging Using CMRD Coated USPIO in the Rat

An MRI scan of a rat taken shortly after administration of 5 mg of CMRDcoated USPIO (Example 43) per kg body weight is shown in FIG. 7B. Theheart, aorta, and coronary artery were found to be readily imaged usingthis agent. An image of the rat taken pre-administration of the agent(FIG. 7A) is included to illustrate the substantial increase in contrasteffected by administration of the test substance.

Example 70 MRI of CMRD Coated USPIO in the Pig

FIG. 8 illustrates enhanced MRI visualization of the heart andsurrounding arteries, as well as the lungs and kidneys of the pig. Fourdoses of 0.4, 0.8, 1.6, and 2.2 mg of iron/kg body weight of sample(Example 43) were each administered to the pig in sequential order. Eachdose was followed by administration of 20 mL of physiological saline,and an MRI image was obtained after each dose. The image shown in FIG.8B is representative of images obtained after each administration. Apre-image of the pig (FIG. 8A) is included to illustrate the substantialincrease in contrast effected by the agent.

A problem associated with low molecular weight gadolinium based contrastagents is that they leak from the vascular space into the interstitialspace and create a hazy background. This hazy background interferes witheffective use of second or third injections of a contrast agentadministered during a single examination. Such extravascular leakagemight not be expected with carboxymethyl reduced dextran-coated USPIOsor similarly prepared alkoxyalkylated polyol or polyether iron oxidecomplexes, due to the relatively large size of the particle, compared tothe size of the particles of a gadolinium contrast agent.

This expectation was confirmed by imaging of rats (Example 69) and inthe data obtained by imaging of the pig (FIG. 8B). No background hazewas observed following use of the CMRD USPIO compositions of the presentinvention. This observation enabled performance of additional vascularimaging tests, after sequential administration of additional doses. Uponintravenous administration, the CMRD coated USPIO, which is anembodiment of the invention, moved as a bolus rapidly into the arteries,organs, and veins, and achieved a uniform distribution in the bloodafter 20 minutes. Upon administration of a second bolus of the agent,additional good images were obtained. A third injection and a fourthinjection were administered with similar results i.e., good images wereobtained. Thus, the process of bolus injection and first passapplication of the CMRD coated USPIO was demonstrated. Further,application of a multiple injection protocol within a reasonably shortperiod of time after the first administration, the entire protocol beingaccomplished in a time period equivalent to a visit by a human subjectto an imaging facility, was also demonstrated.

The principal advantages of capability of multiple bolus injectionswithin a single examination are the opportunities to correct adeficiency in imaging that might arise after an injection, and to imagemultiple parts of the body during a single examination. In this manner,additional sites within the body of a subject can be imaged within ashort period of time after scanning and analysis of earlier images froman earlier pass, and subsequent injections of contrast agent can be usedto obtain different views, or to extend the view in one or more physicaldimensions. For example, detailed analysis of the location and size of ablood clot in a limb such as a leg, can be performed using a series ofviews taken in the each of a first, second, and subsequent passes.

The capability for achieving additional multiple passes ofadministration of a composition of the invention and obtainingadditional rounds of MRI data, beyond a first dose, present strongadvantages of the compositions that are embodiments of the presentinvention. MRI analyses have in the past been limited by the physicallength of the anatomical feature in need of imaging, and by the numbersof structures that can be imaged using a single detection instrumentunit in a given time period.

The results obtained in pigs were observed also in human subjects(Examples 71 and 72).

Example 71 Intravenous Injection of CMRD Coated USPIO into Normal HumanSubjects

The trial design employed thirty-five human subjects each administeredone dose of CMRD T10 coated USPIO prepared according to Example 43(i.v.;1-4 mg of iron/kg body weight). The objectives of Examples 71 and 72were to examine subjects for any potential side effect of the treatment,to obtain data on the composition as an MRI contrast agent, and todetermine the half-life of the composition in blood.

No adverse reactions attributable to administration of the compositionwere observed among the treated subjects at any dose, including thehighest dose (4 mg/kg). For comparison, in clinical trials of FeridexI.V.®, approximately 2-3% of treated patients reported back pain, eventhough Feridex I.V.® and other comparable imaging products areadministered in much smaller doses (e.g., 0.56 mg of iron/kg bodyweight) in order to minimize adverse events and obtain useful contrast.These data indicate that an effective dose of the CMRD coated USPIOparticles of the invention is safer than an effective dose of apreviously approved imaging agent, Feridex I.V.®

Example 72 Rapid Imaging Kinetics and Bio-Distribution in Human Subjects

An initial intravenous bolus injection into human subjects of CMRDcoated USPIO, prepared as in Example 431 yielded a bright MRI of thearterial portion of the circulatory system within 12 secondspost-administration (FIG. 9B). Following a further 15 seconds, MRIexposures yielded bright images of organs and veins. Equilibration ofthe agent throughout the vascular system was achieved within 20 minutes.

The organs capable of being imaged in the early phase followingadministration of the CMRD coated USPIO of the present inventionincluded the heart, arteries and veins. Further, in addition to thelarger elements of the circulatory system, the arterioles and venules ofthe extremities (fingers, toes) could be observed. This level ofresolution allows applications to diagnosis of problems in circulationwithin the extremities, including the detection and localization of anarea of phlebitis. Other organs that were readily imaged include thebrain, kidneys, liver, spleen, and bone marrow. Lymph nodes could beimaged up to several hours after administration of an effective dose.The half-life of the agent in the blood was approximately observed to be10-14 hours (see Table 19 and FIG. 10).

The particles ultimately were removed from circulation by being taken upby the reticuloendothelial system. During the presence of thecomposition at the early phase in the vascular system, and also in thelate or post vascular phase in the reticuloendothelial system (RES),this composition was not observed to enter into interstitial spacesbetween cells. Thus, a hazy background, found to appear with usage ofother compositions, for example, gadolinium based MR contract agentssuch as Magnevist® and DOTOREM®, is avoided during use of the CMRD-USPIOcompositions, as synthesized by the methods of the Examples herein.

TABLE 19 Mean half-life of CMRD-USPIO T10 in human subjects as afunction of dose. Dose mg iron/kg half-life, hours standard deviation #subjects 1 9.7 1.1 8 2 10.3 1.4 8 4 14.4 2.2 17

What is claimed is:
 1. An autoclaved unit dose of an iron oxide complexcomprising: particles of a superparamagnetic iron oxide coated with acarboxymethylated reduced dextran having between about 1100 micromolesand about 1500 micromoles carboxyl per gram of carboxymethylated reduceddextran, wherein the reduced dextran has an average molecular weight ofabout 10 kDa, and wherein the particles have a diameter between about 10nm and about 50 nm, and a biocompatible liquid; wherein the autoclavedunit dose has about 500 mg to about 600 mg of iron.
 2. The autoclavedunit dose of claim 1, wherein the unit dose has less than about 0.1%free iron concentration as determined by atomic absorption spectroscopyon a sample of the unit dose filtered through a 30K molecular weightcutoff micropartition membrane filter.
 3. The autoclaved unit dose ofclaim 1, wherein the particles have a magnetic susceptibility of greaterthan 20,000×10⁻⁶ cgs.
 4. A pharmacological composition comprising: anautoclaved colloid comprising particles of a superparamagnetic ironoxide coated with a carboxymethylated reduced dextran, the reduceddextran having an average molecular weight of about 10 kDa; and abiocompatible liquid, wherein the particles in the autoclaved colloidhaving a diameter between about 10 nm and about 50 nm.
 5. Thepharmacological composition of claim 4, wherein the carboxymethylatedreduced dextran has at least about 1100 micromoles carboxyl per gram ofthe carboxymethylated reduced dextran.
 6. The pharmacologicalcomposition of claim 4, having a magnetic susceptibility of greater than20,000×10⁻⁶ cgs.
 7. The pharmacological composition of claim 4, whereinthe pharmacological composition has less than about 0.1% free iron asdetermined by a dialysis assay.
 8. The pharmacological composition ofclaim 6, wherein the pharmacological composition has a free ironconcentration of less than about 0.1% as determined by atomic absorptionspectroscopy on a sample of the pharmacological composition filteredthrough a 30K molecular weight cutoff micropartition membrane filter. 9.The autoclaved unit dose of claim 1, further comprising mannitol.